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ABSTRACT: Abstract LYVE-1 is a marker expressed by lymphatic endothelial cells (LECs) that line the lymphatic endothelium. Through studies designed to examine potential changes in expression of LYVE-1 in cynomolgus macaque colon tissues during the course of simian immunodeficiency virus (SIV) infection, we discovered that LYVE-1 was expressed by heterogenous populations of cells. As revealed by in situ hybridization (ISH), LYVE-1 mRNA levels in colon were decreased in macaques with AIDS compared with acutely infected or uninfected macaques. In the submucosal layer of the colon, approximately half of the LYVE-1-expressing cells co-expressed the dendritic cell (DC) marker, DC-SIGN/CD209, and this percentage did not change appreciably during infection. Subsets of cells expressing LYVE-1 also co-expressed macrophage markers, such as CD68 and the macrophage mannose receptor (MMR)/CD206, in both the colon and lymph nodes. LECs, DCs, and macrophages that co-expressed LYVE-1 were observed in colon and lymph node from uninfected, healthy animals as well as in tissues with SIV-driven inflammation. These findings provide further definition of the phenotypic overlap between LECs and antigen presenting cells, reveal the heterogeneity within the population of cells expressing the lymphatic marker LYVE-1, and show that SIV modulates this population of cells in a mucosal surface across which the virus is acquired.
Lymphatic Research and Biology 03/2013; 11(1):26-34.
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ABSTRACT: BACKGROUND:: Chemokines provide critical immune cell homing and activation signals that if altered could affect the inflammatory milieu and cellular composition of lymphoid tissues. During HIV-1 and SIV infection, the virus triggers an increase in inflammation/activation, leading to immunodeficiency and development of opportunistic infections, such as in the lungs - a massive interface between the host and the environment. METHODS:: Chemokine, cytokine, and chemokine receptor expression profiles were determined using real-time RT-PCR and in situ hybridization in hilar lymph nodes from cynomolgus macaques at different stages after infection with SIV/DeltaB670. Immunostaining of tissue sections and flow cytometric analysis of cryopreserved cells were used to examine cellular compositions of lymph nodes. RESULTS:: IFN-γ, type 1 chemokine and cognate chemokine receptor mRNAs were up-regulated, whereas type 2 and homeostatic chemokine and chemokine receptor mRNAs were down-regulated in hilar lymph nodes after SIV infection. Local SIV and IFN-γ levels were positively correlated with type 1 chemokine levels, but negatively correlated with type 2 and homeostatic chemokine levels. Using in situ hybridization, Pneumocystis carrini rRNA was detected in lung-draining lymph nodes from animals with P. carrini pneumonia. Changes in the cellular composition of hilar lymph nodes included decreased proportions of CD4 cells and dendritic cells, and increased proportions of CD8, CXCR3 and CCR5 cells. CONCLUSIONS:: SIV infection of cynomolgus macaques dramatically alters the cellular homing signals of lung-draining lymph nodes, which correlated with changes in the immune cellular composition. These changes could contribute to the loss of immune function that defines AIDS.
JAIDS Journal of Acquired Immune Deficiency Syndromes 02/2013; · 4.43 Impact Factor
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ABSTRACT: CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG(2a) fusion protein (fCCL20-mIgG(2a)) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.
Cytokine 01/2013; · 3.02 Impact Factor
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Samantha R Slight,
Javier Rangel-Moreno,
Radha Gopal,
Yinyao Lin, Beth A Fallert Junecko,
Smriti Mehra,
Moises Selman,
Enrique Becerril-Villanueva,
Javier Baquera-Heredia,
Lenin Pavon,
Deepak Kaushal,
Todd A Reinhart,
Troy D Randall,
Shabaana A Khader
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ABSTRACT: One third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Although most infected people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the current challenge is to identify immune parameters that distinguish individuals with latent TB from those with active TB. Using human and experimental models of Mtb infection, we demonstrated that organized ectopic lymphoid structures containing CXCR5+ T cells were present in Mtb-infected lungs. In addition, we found that in experimental Mtb infection models, the presence of CXCR5+ T cells within ectopic lymphoid structures was associated with immune control. Furthermore, in a mouse model of Mtb infection, we showed that activated CD4+CXCR5+ T cells accumulated in Mtb-infected lungs and produced proinflammatory cytokines. Mice deficient in Cxcr5 had increased susceptibility to TB due to defective T cell localization within the lung parenchyma. We demonstrated that CXCR5 expression in T cells mediated correct T cell localization within TB granulomas, promoted efficient macrophage activation, protected against Mtb infection, and facilitated lymphoid follicle formation. These data demonstrate that CD4+CXCR5+ T cells play a protective role in the immune response against TB and highlight their potential use for future TB vaccine design and therapy.
The Journal of clinical investigation 01/2013; · 15.39 Impact Factor
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Samantha Slight,
Javier Rangel-Moreno,
Radha Gopal Yinyao Yin, Beth A. Fallert Junecko,
Smriti Mehra,
Moises Selman,
Enrique Becerril-Villanueva,
Javier Baquera-Heredia,
Lenin Pavon,
Deepak Kaushal,
Todd A. Reinhart,
Troy D. Randall,
and Shabaana A. Khader
[show abstract]
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ABSTRACT: One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Although most infected
people remain asymptomatic, they have a 10% lifetime risk of developing active tuberculosis (TB). Thus, the
current challenge is to identify immune parameters that distinguish individuals with latent TB from those
with active TB. Using human and experimental models of Mtb infection, we demonstrated that organized ectopic
lymphoid structures containing CXCR5+ T cells were present in Mtb-infected lungs. In addition, we found
that in experimental Mtb infection models, the presence of CXCR5+ T cells within ectopic lymphoid structures
was associated with immune control. Furthermore, in a mouse model of Mtb infection, we showed that
activated CD4+CXCR5+ T cells accumulated in Mtb-infected lungs and produced proinflammatory cytokines.
Mice deficient in Cxcr5 had increased susceptibility to TB due to defective T cell localization within the lung
parenchyma. We demonstrated that CXCR5 expression in T cells mediated correct T cell localization within
TB granulomas, promoted efficient macrophage activation, protected against Mtb infection, and facilitated
lymphoid follicle formation. These data demonstrate that CD4+CXCR5+ T cells play a protective role in the
immune response against TB and highlight their potential use for future TB vaccine design and therapy.
The Journal of clinical investigation 01/2013; · 15.39 Impact Factor
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Shabaana A Khader,
Lokesh Guglani,
Javier Rangel-Moreno,
Radha Gopal, Beth A Fallert Junecko,
Jeffrey J Fountain,
Cynthia Martino,
John E Pearl,
Michael Tighe,
Yin-yao Lin,
Samantha Slight,
Jay K Kolls,
Todd A Reinhart,
Troy D Randall,
Andrea M Cooper
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ABSTRACT: IL-23 is required for the IL-17 response to infection with Mycobacterium tuberculosis, but is not required for the early control of bacterial growth. However, mice deficient for the p19 component of IL-23 (Il23a(-/-)) exhibit increased bacterial growth late in infection that is temporally associated with smaller B cell follicles in the lungs. Cxcl13 is required for B cell follicle formation and immunity during tuberculosis. The absence of IL-23 results in decreased expression of Cxcl13 within M. tuberculosis-induced lymphocyte follicles in the lungs, and this deficiency was associated with increased cuffing of T cells around the vessels in the lungs of these mice. Il23a(-/-) mice also poorly expressed IL-17A and IL-22 mRNA. These cytokines were able to induce Cxcl13 in mouse primary lung fibroblasts, suggesting that these cytokines are likely involved in B cell follicle formation. Indeed, IL-17RA-deficient mice generated smaller B cell follicles early in the response, whereas IL-22-deficient mice had smaller B cell follicles at an intermediate time postinfection; however, only Il23a(-/-) mice had a sustained deficiency in B cell follicle formation and reduced immunity. We propose that in the absence of IL-23, expression of long-term immunity to tuberculosis is compromised due to reduced expression of Cxcl13 in B cell follicles and reduced ability of T cells to migrate from the vessels and into the lesion. Further, although IL-17 and IL-22 can both contribute to Cxcl13 production and B cell follicle formation, it is IL-23 that is critical in this regard.
The Journal of Immunology 11/2011; 187(10):5402-7. · 5.79 Impact Factor
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Mingjian Fei,
Shikha Bhatia,
Timothy B. Oriss,
Manohar Yarlagadda,
Anupriya Khare,
Shizuo Akira,
Shinobu Saijo,
Yoichiro Iwakura, Beth A. Fallert Junecko,
Todd A. Reinhart,
Oded Foreman,
Prabir Ray,
Jay Kolls,
Anuradha Ray
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ABSTRACT: Aspergillus fumigatus is commonly associated with allergic bronchopulmonary aspergillosis in patients with severe asthma in which chronic airway
neutrophilia predicts a poor outcome. We were able to recapitulate fungus-induced neutrophilic airway inflammation in a mouse
model in our efforts to understand the underlying mechanisms. However, neutrophilia occurred in a mouse strain-selective fashion,
providing us with an opportunity to perform a comparative study to elucidate the mechanisms involved. Here we show that TNF-α,
largely produced by Ly6c+CD11b+ dendritic cells (DCs), plays a central role in promoting IL-17A from CD4+ T cells and collaborating with it to induce airway neutrophilia. Compared with C57BL/6 mice, BALB/c mice displayed significantly
more TNF-α–producing DCs and macrophages in the lung. Lung TNF-α levels were drastically reduced in CD11c-DTR BALB/c mice
depleted of CD11c+ cells, and TNF-α–producing Ly6c+CD11b+ cells were abolished in Dectin-1−/− and MyD88−/− BALB/c mice. TNF-α deficiency itself blunted accumulation of inflammatory Ly6c+CD11b+ DCs. Also, lack of TNF-α decreased IL-17A but promoted IL-5 levels, switching inflammation from a neutrophil to eosinophil
bias resembling that in C57BL/6 mice. The TNF-αlow DCs in C57BL/6 mice contained more NF-κB p50 homodimers, which are strong repressors of TNF-α transcription. Functionally,
collaboration between TNF-α and IL-17A triggered significantly higher levels of the neutrophil chemoattractants keratinocyte
cytokine and macrophage inflammatory protein 2 in BALB/c mice. Our study identifies TNF-α as a molecular switch that orchestrates
a sequence of events in DCs and CD4 T cells that promote neutrophilic airway inflammation.
Proceedings of the National Academy of Sciences 03/2011; 108(13):5360-5365. · 9.68 Impact Factor
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Mingjian Fei,
Shikha Bhatia,
Timothy B Oriss,
Manohar Yarlagadda,
Anupriya Khare,
Shizuo Akira,
Shinobu Saijo,
Yoichiro Iwakura, Beth A Fallert Junecko,
Todd A Reinhart,
Oded Foreman,
Prabir Ray,
Jay Kolls,
Anuradha Ray
[show abstract]
[hide abstract]
ABSTRACT: Aspergillus fumigatus is commonly associated with allergic bronchopulmonary aspergillosis in patients with severe asthma in which chronic airway neutrophilia predicts a poor outcome. We were able to recapitulate fungus-induced neutrophilic airway inflammation in a mouse model in our efforts to understand the underlying mechanisms. However, neutrophilia occurred in a mouse strain-selective fashion, providing us with an opportunity to perform a comparative study to elucidate the mechanisms involved. Here we show that TNF-α, largely produced by Ly6c(+)CD11b(+) dendritic cells (DCs), plays a central role in promoting IL-17A from CD4(+) T cells and collaborating with it to induce airway neutrophilia. Compared with C57BL/6 mice, BALB/c mice displayed significantly more TNF-α-producing DCs and macrophages in the lung. Lung TNF-α levels were drastically reduced in CD11c-DTR BALB/c mice depleted of CD11c+ cells, and TNF-α-producing Ly6c(+)CD11b(+) cells were abolished in Dectin-1(-/-) and MyD88(-/-) BALB/c mice. TNF-α deficiency itself blunted accumulation of inflammatory Ly6c(+)CD11b(+) DCs. Also, lack of TNF-α decreased IL-17A but promoted IL-5 levels, switching inflammation from a neutrophil to eosinophil bias resembling that in C57BL/6 mice. The TNF-α(low) DCs in C57BL/6 mice contained more NF-κB p50 homodimers, which are strong repressors of TNF-α transcription. Functionally, collaboration between TNF-α and IL-17A triggered significantly higher levels of the neutrophil chemoattractants keratinocyte cytokine and macrophage inflammatory protein 2 in BALB/c mice. Our study identifies TNF-α as a molecular switch that orchestrates a sequence of events in DCs and CD4 T cells that promote neutrophilic airway inflammation.
Proceedings of the National Academy of Sciences 03/2011; 108(13):5360-5. · 9.68 Impact Factor
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Xinmei Zhu, Beth A Fallert-Junecko,
Mitsugu Fujita,
Ryo Ueda,
Gary Kohanbash,
Edward R Kastenhuber,
Heather A McDonald,
Yan Liu,
Pawel Kalinski,
Todd A Reinhart,
Andres M Salazar,
Hideho Okada
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ABSTRACT: Stimulation of double-stranded (ds)RNA receptors can increase the effectiveness of cancer vaccines, but the underlying mechanisms are not completely elucidated. In this study, we sought to determine critical roles of host IFN-alpha and IFN-gamma pathways in the enhanced therapeutic efficacy mediated by peptide vaccines and polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in the murine central nervous system (CNS) GL261 glioma. C57BL/6-background wild type (WT), IFN-alpha receptor-1 (IFN-alphaR1)(-/-) or IFN-gamma(-/-) mice bearing syngeneic CNS GL261 glioma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes with or without intramuscular (i.m.) injections of poly-ICLC. The combinational treatment induced a robust transcription of CXCL10 in the glioma site. Blockade of CXCL10 with a specific monoclonal antibody (mAb) abrogated the efficient CNS homing of antigen-specific type-1 CTL (Tc1). Both IFN-alphaR(-/-) and IFN-gamma(-/-) hosts failed to up-regulate the CXCL10 mRNA and recruit Tc1 cells to the tumor site, indicating non-redundant roles of type-1 and type-2 IFNs in the effects of poly-ICLC-assisted vaccines. The efficient trafficking of Tc1 also required Tc1-derived IFN-gamma. Our data point to critical roles of the host-IFN-alpha and IFN-gamma pathways in the modulation of CNS glioma microenvironment, and the therapeutic effectiveness of poly-ICLC-assisted glioma vaccines.
Cancer Immunology and Immunotherapy 09/2010; 59(9):1401-9. · 3.70 Impact Factor
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ABSTRACT: Infection by HIV-1 frequently leads to pulmonary complications, including alterations to local immune environments. To better understand these alterations, we have examined in detail the patterns and levels of expression of chemokine, cytokine, and chemokine receptor mRNAs in lung tissues from 16 uninfected or simian immunodeficiency virus (SIV)/DeltaB670 infected cynomolgus macaques at different stages of infection. Among the most up-regulated immune genes were interferon (IFN)-gamma, IFN-gamma-inducible CXCR3 ligands, and CCR5 ligands, as well as the cognate chemokine receptors. These changes were greatest in animals with clear Pneumocystis carinii coinfection. Immunohistochemistry and in situ hybridization revealed monocytes/macrophages to be the predominant type of cell infiltrating into lung tissues and serving as the major cellular source of chemokines. To explore the causes of chemokine alterations, we treated macaque lung cells with IFN-gamma, lipopolysaccharide, Poly(I:C), and P. carinii in vitro, and results revealed that these stimuli can induce the expression of CXCR3 ligand and/or CCR5 ligand mRNAs. Taken together, these studies provide a comprehensive definition of the chemokine networks available to modulate cellular recruitment to lung tissues during SIV infection and implicate both cytokines (IFN-gamma) and pathogens (SIV and P. carinii) as contributors to increased expression of pro-inflammatory chemokines.
American Journal Of Pathology 09/2010; 177(3):1274-85. · 4.89 Impact Factor
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Shulin Qin,
Yongjun Sui,
Adam C Soloff, Beth A Fallert Junecko,
Denise E Kirschner,
Michael A Murphey-Corb,
Simon C Watkins,
Patrick M Tarwater,
James E Pease,
Simon M Barratt-Boyes,
Todd A Reinhart
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ABSTRACT: Regulatory T cells (T(reg)) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on T(reg) levels in lymphoid tissues remain incompletely understood. To explore this issue, we have measured the levels of forkhead box protein 3 (FOXP3)-positive cells and associated immunomodulatory genes in a pathogenic simian immunodeficiency virus/macaque model and found that a loss of T(reg) in lymph nodes occurred following simian immunodeficiency virus infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7 and the cytokines TGF-beta and IL-2 were all linked to this loss of T(reg), which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of T(reg), including reduced levels of cytokines associated with T(reg) differentiation and altered expression of agonist and antagonist chemokines. The loss of T(reg) and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1-infected individuals and suggest that components of the T(reg) differentiation and trafficking network could be targets for therapeutic intervention.
The Journal of Immunology 05/2008; 180(8):5530-6. · 5.79 Impact Factor
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ABSTRACT: The lymphatic endothelium is the preferred route for the drainage of interstitial fluid from tissues and also serves as a conduit for peripheral dendritic cells (DCs) to reach draining lymph nodes. Lymphatic endothelial cells (LECs) are known to produce chemokines that recruit Ag-loaded DCs to lymphatic vessels and therefore are likely to regulate the migration of DCs to lymph nodes. TLRs are immune receptors that recognize pathogen associated molecular patterns and then signal and stimulate production of inflammatory chemokines and cytokines that contribute to innate and adaptive immune responses. TLRs are known to be expressed by a wide variety of cell types including leukocytes, epithelial cells, and endothelial cells. Because the TLR expression profile of LECs remains largely unexamined, we have undertaken a comprehensive study of the expression of TLR1-10 mRNAs and protein in primary human dermal (HD) and lung LECs as well as in htert-HDLECs, which display a longer life-span than HDLECs. We found that all three cell types expressed TLR1-6 and TLR9. The responsiveness of these LECs to a panel of ligands for TLR1-9 was measured by real-time RT-PCR, ELISA, and flow cytometry, and revealed that the LECs responded to most but not all TLR ligands by increasing expression of inflammatory chemokines, cytokines, and adhesion molecules. These findings provide insight into the ability of cells of the lymphatic vasculature to respond to pathogens and potential vaccine adjuvants and shape peripheral environments in which DCs will acquire Ag and environmental cues.
The Journal of Immunology 04/2008; 180(5):3399-405. · 5.79 Impact Factor
