Francis H. W. Shand

Cellular and Molecular Mechanisms of Chronic Inflammation-Associated Organ Fibrosis

Organ fibrosis is a pathological condition associated with chronic inflammatory diseases. In fibrosis, excessive deposition of extracellular matrix (ECM) severely impairs tissue architecture and function, eventually resulting in organ failure. This process is mediated primarily by the induction of myofibroblasts, which produce large amounts of collagen I, the main component of the ECM. Accordingly, the origin, developmental pathways, and mechanisms of myofibroblast regulation are attracting increasing attention as potential therapeutic targets. The fibrotic cascade, from initial epithelial damage to eventual myofibroblast induction, is mediated by complex biological processes such as macrophage infiltration, a shift from Th1 to Th2 phenotype, and by inflammatory mediators such as transforming growth factor-β. Here, we review the current understanding of the cellular and molecular mechanisms underlying organ fibrosis.

Myeloid cell population dynamics in healthy and tumor-bearing mice

Tumor growth is often associated with the aberrant systemic accumulation of myeloid-derived suppressor cells (MDSCs), which are a heterogenous population of cells composed of polymorphonuclear neutrophils, monocytes, macrophages, dendritic cells and early myeloid precursors. These MDSCs are thought to suppress anti-tumor T cell responses in both tumor tissues and secondary lymphoid tissues. Accumulation of MDSCs in these target tissues is a dynamic process associated with medullary and extramedullary myelopoiesis and subsequent cellular migration. Here, we review the current understanding of the cellular, molecular, hematological and anatomical principles of MDSC development and migration in tumor-bearing mice. We also discuss the therapeutic potential of chemokines that influence the balance between MDSC subpopulations.

Tracking of intertissue migration reveals the origins of tumor-infiltrating monocytes

Significance Solid tumors contain large numbers of immune cells, including monocytes and monocyte-derived macrophages that promote tumor progression. During tumor development, monocytes accumulate in the spleen. However, the influence of spleen cells on tumor growth remains controversial. Here, we used novel methods for tracking intertissue migration and monitoring hematopoiesis to show that during tumor development the bone marrow dramatically accelerates production of monocytes, rapidly transferring many of these newly formed cells to a reservoir in the spleen. However, these spleen monocytes are less able than their bone marrow counterparts to enter the tumor and make only a minor contribution to the tumor-infiltrating monocyte population. These findings clarify the roles of the spleen and bone marrow in cancer development. Myeloid cells such as monocytes and monocyte-derived macrophages promote tumor progression. Recent reports suggest that extramedullary hematopoiesis sustains a sizable reservoir of tumor-infiltrating monocytes in the spleen. However, the influence of the spleen on tumor development and the extent to which spleen monocytes populate the tumor relative to bone marrow (BM) monocytes remain controversial. Here, we used mice expressing the photoconvertible protein Kikume Green-Red to track the redistribution of monocytes from the BM and spleen, and mice expressing fluorescent ubiquitination-based cell-cycle indicator proteins to monitor active hematopoiesis in these tissues. In mice bearing late-stage tumors, the BM, besides being the major site of monocyte production, supplied the expansion of the spleen reservoir, replacing 9% of spleen monocytes every hour. Deployment of monocytes was equally rapid from the BM and the spleen. However, BM monocytes were younger than those in the spleen and were 2.7 times more likely to migrate into the tumor from the circulation. Partly as a result of this intrinsic difference in migration potential, spleen monocytes made only a minor contribution to the tumor-infiltrating monocyte population. At least 27% of tumor monocytes had traveled from the BM in the last 24 h, compared with only 2% from the spleen. These observations highlight the importance of the BM as the primary hematopoietic tissue and monocyte reservoir in tumor-bearing mice, despite the changes that occur in the spleen monocyte reservoir during tumor development.

Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes

Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103+DCs, including Langerin+CD103+dermal DCs (DDCs), remained in the dLN for 4–4.5 days after migration from the skin, while CD103−DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103−DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103−DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103−DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103−DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells

Memory CD4+ T cells are central regulators of both humoral and cellular immune responses. T cell differentiation results in specific changes in chromatin structure and DNA methylation of cytokine genes. Although the methylation status of a limited number of gene loci in T cells has been examined, the genome-wide DNA methylation status of memory CD4+ T cells remains unexplored. To further elucidate the molecular signature of memory T cells, we conducted methylome and transcriptome analyses of memory CD4+ T cells generated using T cells from TCR-transgenic mice. The resulting genome-wide DNA methylation profile revealed 1144 differentially methylated regions (DMRs) across the murine genome during the process of T cell differentiation, 552 of which were associated with gene loci. Interestingly, the majority of these DMRs were located in introns. These DMRs included genes such as CXCR6, Tbox21, Chsy1, and Cish, which are associated with cytokine production, homing to bone marrow, and immune responses. Methylation changes in memory T cells exposed to specific Ag appeared to regulate enhancer activity rather than promoter activity of immunologically relevant genes. In addition, methylation profiles differed between memory T cell subsets, demonstrating a link between T cell methylation status and T cell differentiation. By comparing DMRs between naive and Ag-specific memory T cells, this study provides new insights into the functional status of memory T cells.

Robust Antitumor Effects of Combined Anti–CD4-Depleting Antibody and Anti–PD-1/PD-L1 Immune Checkpoint Antibody Treatment in Mice

Ueha, Yokochi, Ishiwata, and colleagues show in three mouse tumor models that CD4 depletion led to tumor-specific CTL proliferation in the draining lymph node and increased tumor infiltration of PD-1+CD8+ T cells; it also synergized with PD-1/PD-L1 blockade to suppress tumor growth and prolong survival. Depletion of CD4+ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4+ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti–CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25+ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8+ cell depletion. CD4+ cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti–PD-1 or anti–PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti–PD-1 or anti–PD-L1 mAb therapies. Cancer Immunol Res; 3(6); 631–40. ©2015 AACR.

In vitro and in vivo evidence for anti-inflammatory properties of 2-methoxyestradiol

2-Methoxyestradiol (2MEO) is an endogenous metabolite of 17β-estradiol that interacts with estrogen receptors and microtubules. It has acute anti-inflammatory activity in animal models that is not attributable to known antiproliferative or antiangiogenic actions. Because macrophages are central to the innate inflammatory response, we examined whether suppression of macrophage activation by 2MEO could account for some of its anti-inflammatory effects. Inflammatory mediator production stimulated by lipopolysaccharide (LPS) and interferon-γ in the J774 murine macrophage cell line or human monocytes was measured after treatment with 2MEO or the anti-inflammatory agent dexamethasone. The effect of these agents on LPS-induced acute lung inflammation in mice was also examined. 2MEO suppressed J774 macrophage interleukin-6 and prostaglandin E2 production (by 30 and 47%, respectively, at 10 μM) and human monocyte tumor necrosis factor-α production (by 60% at 3 μM). Estradiol had no effect on J774 macrophage activation, nor did the estrogen receptor antagonist 7α-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17β-diol (ICI 182,780) prevent the effects of 2MEO. The actions of 2MEO were not mimicked by the microtubule-interfering agents colchicine or paclitaxel. In mice exposed to LPS, bronchoalveolar lavage protein content, a measure of vascular leak and epithelial injury, was reduced to a comparable extent (∼54%) by treatment with 2MEO (150 mg · kg−1) or dexamethasone (1 mg · kg−1). In addition, 2MEO reduced LPS-induced interleukin-6 gene expression. Thus, 2MEO modulates macrophage activation in vitro and has high-dose acute anti-inflammatory activity in vivo. These findings are consistent with the acute anti-inflammatory actions of 2MEO being mediated in part by the suppression of macrophage activation.

Loss of Lymph Node Fibroblastic Reticular Cells and High Endothelial Cells Is Associated with Humoral Immunodeficiency in Mouse Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a major risk factor for prolonged humoral immunodeficiency and vaccine unresponsiveness after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the underlying mechanisms for this immunodeficiency are poorly understood. In this article, we describe previously overlooked impacts of GVHD on lymph node (LN) stromal cells involved in humoral immune responses. In major- and minor-mismatched mouse allo-HSCT models, recipients with CD8+ T cell–mediated GVHD suffered severe and irreversible damage to LN structure. These mice were susceptible to pathogenic infection and failed to mount humoral immune responses despite the presence of peripheral T and B cells. These humoral immune defects were associated with the early loss of fibroblastic reticular cells, most notably the CD157+ cell subset, as well as structural defects in high endothelial venules. The disruption to these LN stromal cells was dependent on alloantigens expressed by nonhematopoietic cells. Blockade of the Fas-FasL pathway prevented damage to CD157+ fibroblastic reticular cells and ameliorated LN GVHD. However, blockade of CD62L- or CCR7-dependent migration of CD8+ T cells to the LN was insufficient to prevent stromal cell injury. Overall, our results highlight GVHD-associated loss of functional stromal cells and LN GVHD as a possible explanation for the prolonged susceptibility to infectious disease that is experienced by allo-HSCT patients.

A rare subset of skin-tropic regulatory T cells expressing Il10 / Gzmb inhibits the cutaneous immune response

Foxp3+ regulatory T cells (Tregs) migrating from the skin to the draining lymph node (dLN) have a strong immunosuppressive effect on the cutaneous immune response. However, the subpopulations responsible for their inhibitory function remain unclear. We investigated single-cell gene expression heterogeneity in Tregs from the dLN of inflamed skin in a contact hypersensitivity model. The immunosuppressive genes Ctla4 and Tgfb1 were expressed in the majority of Tregs. Although Il10-expressing Tregs were rare, unexpectedly, the majority of Il10-expressing Tregs co-expressed Gzmb and displayed Th1-skewing. Single-cell profiling revealed that CD43+ CCR5+ Tregs represented the main subset within the Il10/Gzmb-expressing cell population in the dLN. Moreover, CD43+ CCR5+ CXCR3− Tregs expressed skin-tropic chemokine receptors, were preferentially retained in inflamed skin and downregulated the cutaneous immune response. The identification of a rare Treg subset co-expressing multiple immunosuppressive molecules and having tissue-remaining capacity offers a novel strategy for the control of skin inflammatory responses.

Laryngeal mucus hypersecretion is exacerbated after smoking cessation and ameliorated by glucocorticoid administration.

INTRODUCTION The mechanisms underlying the effects of cigarette smoke and smoking cessation on respiratory secretion, especially in the larynx, remain unclear. OBJECTIVES The aims of this study were to determine the effects of cigarette smoke and smoking cessation on laryngeal mucus secretion and inflammation, and to investigate the effects of glucocorticoid administration. METHODS We administered cigarette smoke solution (CSS) to eight-week-old male Sprague Dawley rats for four weeks, then examined laryngeal mucus secretion and inflammatory cytokine expression on days 1, 28 and 90 after smoking cessation. We also investigated the effects of the glucocorticoid triamcinolone acetonide when administered on day 1 after smoking cessation. RESULTS Exposure to CSS resulted in an increase in laryngeal mucus secretion that was further excacerbated following smoking cessation. This change coincided with an increase in the expression of mRNA for the inflammatory cytokines tumor necrosis factor and interleukin-6, as well as mRNA for MUC5AC, which is involved in mucin production. Triamcinolone suppressed CSS-induced laryngeal mucus hypersecretion and pro-inflammatory cytokine production. CONCLUSION Cigarette smoke-associated inflammation may contribute to the exacerbated laryngeal mucus hypersecretion that occurs following smoking cessation. The inflammatory response represents a promising target for the treatment of cigarette smoke-associated mucus hypersecretion.