Shulin Qin

Human Lymphatic Endothelial Cells Express Multiple Functional TLRs

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.

Innate Stat3-mediated induction of the antimicrobial protein Reg3γ is required for host defense against MRSA pneumonia

STAT3-mediated induction of Reg3γ enhances bacteriostatic and bactericidal activity to pulmonary Staphylococcus aureus.

Chemokine and Cytokine Mediated Loss of Regulatory T Cells in Lymph Nodes during Pathogenic Simian Immunodeficiency Virus Infection

Regulatory T cells (Treg) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on Treg 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 Treg in lymph nodes occurred following simian immunodeficiency virus infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7 and the cytokines TGF-β and IL-2 were all linked to this loss of Treg, which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of Treg, including reduced levels of cytokines associated with Treg differentiation and altered expression of agonist and antagonist chemokines. The loss of Treg and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1-infected individuals and suggest that components of the Treg differentiation and trafficking network could be targets for therapeutic intervention.

The dual role of dendritic cells in the immune response to human immunodeficiency virus type 1 infection

Many aspects of the complex interaction between human immunodeficiency virus type 1 (HIV-1) and the human immune system remain elusive. Our objective was to study these interactions, focusing on the specific roles of dendritic cells (DCs). DCs enhance HIV-1 infection processes as well as promote an antiviral immune response. We explored the implications of these dual roles. A mathematical model describing the dynamics of HIV-1, CD4+ and CD8+ T-cells, and DCs interacting in a human lymph node was analysed and is presented here. We have validated the behaviour of our model against non-human primate simian immunodeficiency virus experimental data and published human HIV-1 data. Our model qualitatively and quantitatively recapitulates clinical HIV-1 infection dynamics. We have performed sensitivity analyses on the model to determine which mechanisms strongly affect infection dynamics. Sensitivity analysis identifies system interactions that contribute to infection progression, including DC-related mechanisms. We have compared DC-dependent and -independent routes of CD4+ T-cell infection. The model predicted that simultaneous priming and infection of T cells by DCs drives early infection dynamics when activated T-helper cell numbers are low. Further, our model predicted that, while direct failure of DC function and an indirect failure due to loss of CD4+ T-helper cells are both significant contributors to infection dynamics, the former has a more significant impact on HIV-1 immunopathogenesis.

Epigallocatechin-3-Gallate Reduces Airway Inflammation in Mice through Binding to Proinflammatory Chemokines and Inhibiting Inflammatory Cell Recruitment

One major activity of chemokines is the recruitment of immune cells to sites of infection and inflammation. CD4+ Th1 cells play critical roles in host defense against pathogens and in the pathogenesis of many immune-mediated diseases. It was reported that epigallocatechin-3-gallate (EGCG) exhibits anti-inflammatory properties, but the mechanisms have not been completely defined. In this study, we found that EGCG markedly decreased recruitment of murine OVA-specific Th1 cells and other inflammatory cells into the airways in a Th1 adoptive-transfer mouse model. In vitro analysis revealed that EGCG inhibited CXCR3 ligand-driven chemotaxis of murine and human cells. Surface plasmon resonance studies revealed that EGCG bound directly to chemokines CXCL9, CXCL10, and CXCL11. These results indicated that one anti-inflammatory mechanism of EGCG is binding of proinflammatory chemokines and limiting their biological activities. These findings support further development of EGCG as a potent therapeutic for inflammatory diseases.

Simian Immunodeficiency Virus Infection Alters Chemokine Networks in Lung Tissues of Cynomolgus Macaques: Association with Pneumocystis carinii Infection

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.

Association between decreased CXCL12 and CCL25 expression and increased apoptosis in lymphoid tissues of cynomolgus macaques during SIV infection

Background  Chemokines likely play multiple roles in HIV‐1 and SIV pathogenesis. To examine potential associations between chemokine expression levels and apoptosis of cells in lymphoid tissues during SIV infection, we measured chemokine and cytokine mRNA levels in multiple lymphoid tissues compartments from uninfected and SIV‐infected cynomolgus macaques (Macaca fascicularis).

Multiple roles for chemokines in the pathogenesis of SIV infection.

Chemokines are small chemoattractant cytokines involved in homeostatic and inflammatory immune cell migration. These small proteins have multiple functional properties that extend beyond their most recognized role in controlling cellular migration. The complex immunobiology of chemokines, coupled with the use of subsets of chemokine receptors as HIV-1 and SIV entry co-receptors, suggests that these immunomodulators could play important roles in the pathogenesis associated with infection by HIV-1 or SIV. This review provides an overview of the effects of pathogenic infection on chemokine expression in the SIV/macaque model system, and outlines potential mechanisms by which changes in these expression profiles could contribute to development of disease. Key challenges faced in studying chemokine function in vivo and new opportunities for further study and development of therapeutic interventions are discussed. Continued growth in our understanding of the effects of pathogenic SIV infection on chemokine expression and function and the continuing development of chemokine receptor targeted therapeutics will provide the tools and the systems necessary for future studies of the roles of chemokines in HIV-1 pathogenesis.

Functional Characterization of Ferret CCL20 and CCR6 and Identification of Chemotactic Inhibitors

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.

Macaque Paneth Cells Express Lymphoid Chemokine CXCL13 and Other Antimicrobial Peptides Not Previously Described as Expressed in Intestinal Crypts

ABSTRACT CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), β-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.