Eun Jeong Park

Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target.

Cyclin D1 (CyD1) is a pivotal cell cycle–regulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also strongly up-regulated at sites of inflammation, its exact roles in this context remain uncharacterized. To address this question, we developed a strategy for selectively silencing CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1–small interfering RNA (siRNA). Antibodies to β7 integrin (β7 I) were then used to target specific leukocyte subsets involved in gut inflammation. Systemic application of β7 I-tsNPs silenced CyD1 in leukocytes and reversed experimentally induced colitis in mice by suppressing leukocyte proliferation and T helper cell 1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings.

Intracellularly Expressed TLR2s and TLR4s Contribution to an Immunosilent Environment at the Ocular Mucosal Epithelium

Epithelial cells are key players in the first line of defense offered by the mucosal immune system against invading pathogens. In the present study we sought to determine whether human corneal epithelial cells expressing Toll-like receptors (TLRs) function as pattern-recognition receptors in the innate immune system and, if so, whether these TLRs act as a first line of defense in ocular mucosal immunity. Incubation of human primary corneal epithelial cells and the human corneal epithelial cell line (HCE-T) with peptidoglycan or LPS did not lead to activation, at the level of DNA transcription, of NF-κB or the secretion of inflammation-associated molecules such as IL-6, IL-8, and human β-defensin-2. However, when incubated with IL-1α to activate NF-κB, the production by these cells of such inflammatory mediators was enhanced. Human corneal epithelial cells were observed to express both TLR2- and TLR4-specific mRNA as well as their corresponding proteins intracellularly, but not at the cell surface. However, even when LPS was artificially introduced into the cytoplasm, it did not lead to the activation of epithelial cells. Taken together, our results demonstrate that the intracellular expression of TLR2 and TLR4 in human corneal epithelial cells fails to elicit innate immune responses and therefore, perhaps purposely, contributes to an immunosilent environment at the ocular mucosal epithelium.

Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes

During the course of homing to lymph nodes (LNs), T cells undergo a multistep adhesion cascade that culminates in a lymphocyte function-associated antigen 1 (LFA-1)-dependent firm adhesion to the luminal surface of high endothelial venules (HEVs). The importance of LFA-1 affinity regulation in supporting T-cell arrest on HEVs has been well established, however, its importance in the postadhesion phase, which involves intraluminal crawling and diapedesis to the extravascular space, remains elusive. Here we have shown that LFA-1 affinity needs to be appropriately regulated to support these essential steps in the homing cascade. Genetically engineered T cells that were unable to properly down-regulate LFA-1 affinity underwent enhanced, chemokine-independent arrest in HEVs but showed perturbed intravascular crawling to transmigration sites and compromised diapedesis across HEVs. By contrast, the extravascular migration of T cells was insensitive to the affinity-enhancing LFA-1 mutation. These results highlight the requirement for balanced LFA-1 affinity regulation in intravascular and transvascular, but not extravascular, T-cell migration in LNs.

Advances in understanding sepsis

Summary Sepsis, a systemic inflammatory response to infection, is a leading cause of death in intensive care units. Recent investigations into the pathogenesis of sepsis reveal a biphasic inflammatory process. An early phase is characterized by pro‐inflammatory cytokines (e.g. tumour necrosis factor‐&agr;), whereas a late phase is mediated by an inflammatory high‐mobility group box 1 and an anti‐inflammatory interleukin‐10. Inflammation aberrantly activates coagulation cascades as sepsis progresses. This dual inflammatory response concomitant with dysregulated coagulation partially accounts for unsuccessful anti‐cytokine therapies that have solely targeted early pro‐inflammatory mediators (e.g. tumour necrosis factor‐&agr;). In contrast, activated protein C, which modifies both inflammatory and coagulatory pathways, has improved survival in patients in severe sepsis. Inhibition of the late mediator high‐mobility group box 1 improves survival in established sepsis in pre‐clinical studies. In addition, recent advances in molecular medicine have shed light on two novel experimental interventions against sepsis. Accelerated apoptosis of lymphocytes has been shown to play an important role in organ dysfunction in sepsis and techniques to suppress apoptosis have improved survival rate in sepsis models. The vagus nerve system has also been shown to suppress innate immune response through endogenous release and exogenous administration of cholinergic agonists, ameliorating inflammation and lethality in sepsis models.

Aberrant activation of integrin α4β7 suppresses lymphocyte migration to the gut

Integrin adhesion molecules mediate lymphocyte migration and homing to normal and inflamed tissues. While the ligand-binding activity of integrins is known to be modulated by conformational changes, little is known about how the appropriate balance of integrin adhesiveness is maintained in order to optimize the migratory capacity of lymphocytes in vivo. In this study we examined the regulation of the gut homing receptor alpha4beta7 integrin by manipulating at the germline level an integrin regulatory domain known as adjacent to metal ion-dependent adhesion site (ADMIDAS). ADMIDAS normally serves to raise the activation threshold of alpha4beta7, thereby stabilizing it in the default nonadhesive state. Lymphocytes from knockin beta7 (D146A) mice, which harbor a disrupted ADMIDAS, not only expressed an alpha4beta7 integrin that persistently adhered to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), but also exhibited perturbed cell migration along MAdCAM-1 substrates resulting from improper de-adhesion of the lymphocyte trailing edge. In vivo, aberrantly activated alpha4beta7 enhanced adhesion to Peyers patch venules, but suppressed lymphocyte homing to the gut, diminishing the capacity of T cells to induce colitis. Our results underscore the importance of a proper balance in the adhesion and de-adhesion of the alpha4beta7 integrin, both for lymphocyte trafficking to the gut and for colitis progression.

Cutting edge: Uniqueness of lymphoid chemokine requirement for the initiation and maturation of nasopharynx-associated lymphoid tissue organogenesis.

CD3−CD4+CD45+ inducer cells are required for the initiation of mucosa-associated organogenesis of both nasopharynx-associated lymphoid tissues (NALT) and Peyer’s patches (PP) in the aerodigestive tract. CXCL13−/− mice and mice carrying the paucity of lymph node T cell (plt) mutation and lacking expression of CCL19 and CCL21 accumulate CD3−CD4+CD45+ cells at the site of NALT but not of PP genesis. Although NALT was observed to develop in adult CXCL13−/− and plt/plt mice, the formation of germinal centers in CXCL13−/− mice was affected, and their population of B cells was much lower than in the NALT of CXCL13+/− mice. Similarly, fewer T cells were observed in the NALT of plt/plt mice than in control mice. These findings indicate that the initiation of NALT organogenesis is independent of CXCL13, CCL19, and CCL21. However, the expression of these lymphoid chemokines is essential for the maturation of NALT microarchitecture.

Prenatal Blockage of Lymphotoxin β Receptor and TNF Receptor p55 Signaling Cascade Resulted in the Acceleration of Tissue Genesis for Isolated Lymphoid Follicles in the Large Intestine

Signaling by lymphotoxin (LT) and TNF is essential for the organogenesis of secondary lymphoid tissues in systemic and mucosal compartments. In this study, we demonstrated that the progeny of mice treated with fusion protein of LTβR and IgGFc (LTβR-Ig) or LTβR-Ig plus TNFR55-Ig (double Ig) showed significantly increased numbers of isolated lymphoid follicles (ILF) in the large intestine. Interestingly, double Ig treatment accelerated the maturation of large intestinal ILF. Three-week-old progeny of double Ig-treated mice showed increased numbers of ILF in the large intestine, but not in the small intestine. Furthermore, alteration of intestinal microflora by feeding of antibiotic water did not affect the increased numbers of ILF in the large intestine of double Ig-treated mice. Most interestingly, mice that developed numerous ILF also had increased levels of activation-induced cytidine deaminase expression and numbers of IgA-expressing cells in the lamina propria of the large intestine. Taken together, these results suggest that ILF formation in the large intestine is accelerated by blockage of LTβR and TNFR55 signals in utero, and ILF, like colonic patches, might play a role in the induction of IgA response in the large intestine.

Autocrine IL-15 Mediates Intestinal Epithelial Cell Death Via the Activation of Neighboring Intraepithelial NK Cells

Intestinal intraepithelial lymphocytes (IELs), which reside between the basolateral faces of intestinal epithelial cells (IECs), provide a first-line defense against pathogens via their cytotoxic activity. Although IEC-derived IL-7 and IL-15 are key regulatory cytokines for the development and activation of IELs, we report here that IL-15 but not IL-7 mediates the reciprocal interaction between IELs and IECs, an important interaction for the regulation of appropriate mucosal immunohomeostasis. IL-15-treated IELs induced cell death in IECs via the cytotoxic activity in vitro. Among the different subsets of IL-15-treated IELs, CD4−CD8−TCR− IELs, which express NK marker (DX5 or NK1.1), showed the most potent syngenic IEC killing activity. These intraepithelial NK cells expressed Ly-49 molecules, NKG2 receptors, and perforin. These results suggest the possibility that the cell death program of IECs could be regulated by self-produced IL-15 through the activation of intraepithelial NK cells.

Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [18F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [18F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Clonal Expansion of Double-Positive Intraepithelial Lymphocytes by MHC Class I-Related Chain A Expressed in Mouse Small Intestinal Epithelium

Expression of a distant homologue MHC class I molecule, MHC class I-related chain A (MICA), has been found to be stress inducible and limited to the intestinal epithelium. This nonclassical MHC molecule is associated with various carcinomas in humans. To understand the biological consequences of MICA expression in the gut, we generated transgenic (Tg) mice (T3b-MICA Tg) under the control of the T3b promoter. The T3b-MICA Tg mice expressed MICA selectively in the intestine and had an increased number of TCRαβ CD4CD8αα, double-positive (DP) intraepithelial lymphocytes (IELs) in the small bowel. These MICA-expanded DP IELs exhibited a bias to Vβ8.2 and overlapped motifs of the complementarity-determining region 3 region among various Tg mice. Hence, the overexpression of MICA resulted in a clonal expansion of DP IELs. Studies in model of inflammatory bowel disease showed that transgenic MICA was able to attenuate the acute colitis induced by dextran sodium sulfate administration. Therefore, this unique in vivo model will enable investigation of possible influences of stress-inducible MICA on the gut immune surveillance.