Role of a novel immune modulating DDR2-expressing population in silica-induced pulmonary fibrosis.

Micro-injuries associated with chronic inhaled particle exposures are linked with activation of the immune response and are thought to contribute to progression of fibrotic disease. In the pulmonary environment, we have previously demonstrated a heterogeneous population of circulating fibroblast precursors (CFPs), which are defined by expression of the pan-leukocyte marker CD45 and the collagen receptor, discoidin domain receptor-2 (DDR2). This population is derived from the hematopoietic stem cell, expresses collagen, and has a fibroblastic morphology in vitro. Herein, we demonstrate a novel subset of CFPs expressing immune markers CD11b, CD11c, and major histocompatibility complex II (MHC II). The CFP population was skewed toward this immune marker expressing subset in animals with silica-induced pulmonary fibrosis. Data indicate that this CFP subset upregulates co-stimulatory molecules and MHC II expression in response to silica-induced fibrosis in vivo. Functionally, this population was shown to promote T cell skewing away from a Th1 response and toward a pro-inflammatory profile. These studies represent the first direct flow cytometric and functional evaluation of the novel immune marker expressing CFP subset in an exposure-induced model of pulmonary fibrosis. Elucidating the role of this CFP subset may enhance our understanding of the complex immune balance critical to mediating exposures at the pulmonary-host interface and may be a valuable target for the treatment of exposure-induced pulmonary fibrosis.

Indomethacin Treatment of Mice with Premalignant Oral Lesions Sustains Cytokine Production and Slows Progression to Cancer.

Current treatment options for head and neck squamous cell carcinoma (HNSCC) patients are often ineffective due to tumor-localized and systemic immunosuppression. Using the 4-NQO mouse model of oral carcinogenesis, this study showed that premalignant oral lesion cells produce higher levels of the immune modulator, PGE2, compared to HNSCC cells. Inhibiting prostaglandin production of premalignant lesion cells with the pan-cyclooxygenase inhibitor indomethacin stimulated their induction of spleen cell cytokine production. In contrast, inhibiting HNSCC prostaglandin production did not stimulate their induction of spleen cell cytokine production. Treatment of mice bearing premalignant oral lesions with indomethacin slowed progression of premalignant oral lesions to HNSCC. Flow cytometric analysis of T cells in the regional lymph nodes of lesion-bearing mice receiving indomethacin treatment showed an increase in lymph node cellularity and in the absolute number of CD8+ T cells expressing IFN-γ compared to levels in lesion-bearing mice receiving diluent control treatment. The cytokine-stimulatory effect of indomethacin treatment was not localized to regional lymph nodes but was also seen in the spleen of mice with premalignant oral lesions. Together, these data suggest that inhibiting prostaglandin production at the premalignant lesion stage boosts immune capability and improves clinical outcomes.

Premalignant Oral Lesion Cells Elicit Increased Cytokine Production and Activation of T-cells.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) are known to evade the host immune response. How premalignant oral lesions modulate the immune response, however, has yet to be elucidated. MATERIALS AND METHODS: A mouse model of oral carcinogenesis was used to determine how mediators from premalignant oral lesion cells vs. HNSCC cells impact on immune cytokine production and activation. RESULTS: Media conditioned by premalignant lesion cells elicited an increased production of T cell-associated cytokines and proinflammatory mediators from cervical lymph node cells compared to media conditioned by HNSCC cells or media alone. In the presence of premalignant lesion cell-conditioned media, CD4(+) T cell expression of the IL-2 receptor CD25 and CD8(+) T cell expression of the activation marker CD69 was greater, compared to what was induced in HNSCC cell-conditioned media or media alone. CONCLUSION: Premalignant lesion cells promote a proinflammatory environment and induce immune changes before HNSCC tumors are established.

Treatment to sustain a Th17-type phenotype to prevent skewing toward Treg and to limit premalignant lesion progression to cancer.

While immune suppression is a hallmark of head and neck squamous cell carcinoma (HSNCC), the immunological impact of premalignant oral lesions, which often precedes development of HNSCC, is unknown. The present study assessed the changes in splenic and draining lymph node CD4(+) cell populations and their production of select cytokines that occur in mice with carcinogen-induced premalignant oral lesions and the changes that occur as lesions progress to oral cancer. These studies found skewing toward Th1 and Th17-type phenotypes in the spleen and lymph nodes of mice with premalignant oral lesions and a shift to Treg as lesions progress to cancer. Since the role of Th17 cells in the progression from premalignant lesions to cancer is not clear, studies determined the immunological and clinical effect of treating mice bearing premalignant oral lesions with a TGF-ß type 1 receptor inhibitor plus IL-23 as an approach to sustain the Th17 phenotype. These studies showed that the treatment approach not only sustained the Th17 phenotype, but also increased distal spleen cell and regional lymph node cell production of other stimulatory/inflammatory mediators and slowed premalignant lesion progression to cancer.

Cytokine and Adipokine Levels in Patients with Premalignant Oral Lesions or in Patients with Oral Cancer Who Did or Did Not Receive 1α,25-Dihydroxyvitamin D3 Treatment upon Cancer Diagnosis.

Differences in levels of inflammation-modulating cytokines and adipokines in patients with premalignant oral lesions versus in patients that develop squamous cell carcinoma of the head and neck (HNSCC) were assessed. Also assessed was the impact of treating HNSCC patients with the immune regulatory mediator, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], on modulators of inflammation. Compared to healthy controls, patients with premalignant oral lesions had increases in their systemic levels of the inflammatory cytokines IL-6 and IL-17, and increases in the adipokine, leptin. However, levels of these pro-inflammatory cytokines and adipokine were reduced in patients with HNSCC. Treatment of HNSCC patients with 1,25(OH)2D3 increased levels of each of the measured immune mediators. Levels of the anti-inflammatory adipokine, adiponectin, were shifted inversely with the levels of the pro-inflammatory cytokines and with leptin. These studies demonstrate heightened immune reactivity in patients with premalignant lesions, which wanes in patients with HNSCC, but which is restored by treatment with 1,25(OH)2D3.

An Inflammatory Cytokine Milieu is Prominent in Premalignant Oral Lesions, but Subsides when Lesions Progress to Squamous Cell Carcinoma.

While head and neck squamous cell carcinomas (HNSCC) are associated with profound immune suppression, less is known about the immunological milieu of premalignant oral lesions. The present study shows dynamic shifts in the immune milieu within premalignant oral lesions and when they have progressed to HNSCC. Specifically, this study showed that the premalignant lesion environment consists of inflammatory mediators and IL-17, but this inflammatory phenotype declines when premalignant oral lesions have progressed to HNSCC. The cytokine profiles of human tissues did not correspond with plasma cytokine profiles. A murine carcinogen-induced premalignant lesion model that progresses to HNSCC was used to examine cytokine profiles released from tissues as well as regional lymph nodes. As in human tissues, murine premalignant lesions and regional lymph nodes released high levels of inflammatory cytokines and, very prominently, IL-17. Also similar to human tissues, release of inflammatory cytokines declined in HNSCC tissues of mice and in the regional lymph nodes of mice with HNSCC. Studies focusing on IL-17 showed that mediators from premalignant lesions stimulated normal spleen cells to produce increased levels of IL-17, while mediators from HNSCC were less stimulatory toward IL-17 production. IL-17 production by Th17-skewed CD4+ cells was strongly inhibited by normal oral epithelium as well as HNSCC. In contrast, premalignant lesion-derived mediators further increased IL-17 production by Th17-skewed cells. The stimulation of IL-17 production by premalignant lesions was dependent on IL-23, which premalignant lesions released in higher amounts than control tissues or HNSCC. HNSCC tissues instead produced increased levels of TGF-ß compared to premalignant lesions, and skewed normal spleen cells toward the Treg phenotype. This skewing was blocked by supplementation with IL-23. These studies suggest IL-23 to be a significant contributor to the inflammatory IL-17 phenotype in premalignant oral lesions and suggest the decline in IL-23 in HNSCC leads to a decline in Th17 cells.

Effect of the premalignant and tumor microenvironment on immune cell cytokine production in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is marked by immunosuppression, a state in which the established tumor escapes immune attack. However, the impact of the premalignant and tumor microenvironments on immune reactivity has yet to be elucidated. The purpose of this study was to determine how soluble mediators from cells established from carcinogen-induced oral premalignant lesions and HNSCC modulate immune cell cytokine production. It was found that premalignant cells secrete significantly increased levels of G-CSF, RANTES, MCP-1, and PGE2 compared to HNSCC cells. Splenocytes incubated with premalignant supernatant secreted significantly increased levels of Th1-, Th2-, and Th17-associated cytokines compared to splenocytes incubated with HNSCC supernatant. These studies demonstrate that whereas the premalignant microenvironment elicits proinflammatory cytokine production, the tumor microenvironment is significantly less immune stimulatory and may contribute to immunosuppression in established HNSCC.