Probes for INS

In the present study, we evaluated expression of IFN-γ, IL-17, TNF-α, IL-10 and TGF-β by mucosal cells, including WC1+ γδ T cells, in ileal tissues taken from non-infected cattle and cattle naturally infected with Mycobacterium avium subsp paratuberculosis (MAP). Infected cattle were either in the subclinical or clinical stage of infection. We hypothesized that the cytokine profile of the WC1+ γδ T cell subset would be different between subclinical and clinical cattle. Our data indicate a significant increase in the numbers of WC1+ γδ T cells expressing IL-10 in clinical cattle compared to subclinical and non-infected cattle. We observed a significant increase in TGF-β expression by non-WC1+ cells in clinically infected cattle. Expression of IFN-γ, IL-17 and TNF-α in mucosal cells, including the WC1+ γδ T cell subset, was identified in all examined groups. However, our data indicate that the stage of infection did not significantly influence expression of these proinflammatory cytokines. This study demonstrates changes in the cytokine mRNA expression profile of mucosal cells in the ileum, and specifically WC1+ γδ T cells, as cattle progress to the clinical disease. The change is characterized by an increase in expression of anti-inflammatory cytokines.

Inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), are elevated in ovarian cancer. Differences in cytokine expression by histologic subytpe or ovarian cancer risk factors can provide useful insight into ovarian cancer risk and etiology. We used ribonucleic acid (RNA) in-situ hybridization to assess TNF-α and IL-6 expression on tissue microarray slides from 78 epithelial ovarian carcinomas (51 serous, 12 endometrioid, 7 clear cell, 2 mucinous, 6 other) from a population-based case control study. Cytokine expression was scored semi-quantitatively and odds ratios (OR) and 95% confidence intervals (CI) were calculated using polytomous logistic regression. TNF-α was expressed in 46% of the tumors while sparse IL-6 expression was seen only 18% of the tumors. For both markers, expression was most common in high grade serous carcinomas followed by endometrioid carcinomas. Parity was associated with a reduced risk of TNF-α positive (OR = 0.3, 95% CI: 0.1-0.7 for 3 or more children versus none) but not TNF-α negative tumors (p-heterogeneity = 0.02). In contrast, current smoking was associated with a nearly three fold increase in risk of TNF-α negative (OR = 2.8, 95% CI: 1.2, 6.6) but not TNF-α positive tumors (p-heterogeneity = 0.06). Our data suggests that TNF-α expression in ovarian carcinoma varies by histologic subtype and provides some support for the role of inflammation in ovarian carcinogenesis. The novel associations detected in our study need to be validated in a larger cohort of patients in future studies.

MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples (n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.

The cytokine interleukin-1β (IL-1β) is known to stimulate proinflammatory immune responses and impair β-cell function and viability, all critical events in the pathogenesis of type 1 diabetes (T1D). Here we evaluate the effect of SER140, a small peptide IL-1β receptor antagonist, on diabetes progression and cellular pancreatic changes in female nonobese diabetic (NOD) mice. Eight weeks of treatment with SER140 reduced the incidence of diabetes by more than 50% compared with vehicle, decreased blood glucose, and increased plasma insulin. Additionally, SER140 changed the endocrine and immune cells dynamics in the NOD mouse pancreas. Together, the data suggest that SER140 treatment postpones the onset of diabetes in female NOD mice by interfering with IL-1β activated pathways.

Regardless of host, pathogenic mycobacteria of the Mycobacterium tuberculosiscomplex such as Mycobacterium bovis, induce a characteristic lesion known as agranuloma, tubercle or tuberculoid granuloma. Granulomas represent a distinct host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathogens such as M. bovis. Granulomas are composed of specific cell types including epithelioid macrophages, lymphocytes and a morphologically distinctive cell type, the multinucleated giant cell. Multinucleated giant cells are formed by the fusion of multiple macrophages; however, their function remains unclear. In humans, giant cells in tuberculous granulomas have been shown to express various cytokines, chemokines and enzymes important to the formation and maintenance of the granuloma. The objective of this study was to quantitatively assess multinucleated giant cell cytokine expression in bovine tuberculoid granulomas; focusing on cytokines of suspected relevance to bovine tuberculosis. Using calves experimentally infected with M. bovis, in situ cytokine expression was quantitatively assessed using RNAScope^® for the following cytokines TNF-α, IFN-γ, TGF-β, IL-17A and IL-10. Multinucleated giant cells in bovine tuberculoid granulomas expressed all examined cytokines to varying degrees, with differential expression of TGF-β, IL-17A and IL-10 in giant cells from early versus late stage granulomas. There was a modest, positive correlation between the level of cytokine expression and cell size or number of nuclei. These results suggest that multinucleated giant cells are active participants within bovine tuberculoid granulomas, contributing to the cytokine milieu necessary to form and maintain granulomas.

The hallmark lesion of tuberculosis in humans and animals is the granuloma. The granuloma represents a distinct host cellular immune response composed of epithelioid macrophages, lymphocytes, and multinucleated giant cells, often surrounding a caseous necrotic core. Within the granuloma, host-pathogen interactions determine disease outcome. Factors within the granulomas such as cytokines and chemokines drive cell recruitment, activity, function and ultimately the success or failure of the host's ability to control infection. Hence, an understanding of the granuloma-level cytokine response is necessary to understand tuberculosis pathogenesis. In-situ cytokine expression patterns were measured using a novel in situ hybridization assay, known as RNAScope^® in granulomas of the lungs, tracheobronchial lymph nodes and caudal mediastinal lymph nodes of cattle experimentally infected with Mycobacterium bovis via aerosol exposure. In spite of microscopic morphological similarities, significant differences were seen between late stage granulomas of the lung compared to those of the tracheobronchial lymph nodes for IL-17A, IFN-γ, TGF-β, IL10 and IL-22 but not for TNF-α. Additionally, significant differences were noted between granulomas from two different thoracic lymph nodes that both receive afferent lymphatics from the lungs (i.e., tracheobronchial and caudal mediastinal lymph nodes) for TNF-α, IL-17A, IFN-γ, TGF-β and IL-10 but not for IL-22. These findings show that granuloma morphology alone is not a reliable indicator of granuloma function as granulomas of similar morphologies can have disparate cytokine expression patterns. Moreover, anatomically distinct lymph nodes (tracheobronchial vs caudal mediastinal) differ in cytokine expression patterns even when both receive afferent lymphatics from a lung containing tuberculoid granulomas. These findings show that selection of tissue and anatomic location are critical factors in assessing host immune response to M. bovis and should be considered carefully.

CD4+ T cells that express the forkhead box P3 (FOXP3) transcription factor function as regulatory T (Treg) cells and hinder effective immune responses against cancer cells. Abundant Treg cell infiltration into tumors is associated with poor clinical outcomes in various types of cancers. However, the role of Treg cells is controversial in colorectal cancers (CRCs), in which FOXP3+ T cell infiltration indicated better prognosis in some studies. Here we show that CRCs, which are commonly infiltrated by suppression-competent FOXP3hi Treg cells, can be classified into two types by the degree of additional infiltration of FOXP3lo nonsuppressive T cells. The latter, which are distinguished from FOXP3+ Treg cells by non-expression of the naive T cell marker CD45RA and instability of FOXP3, secreted inflammatory cytokines. Indeed, CRCs with abundant infiltration of FOXP3lo T cells showed significantly better prognosis than those with predominantly FOXP3hi Treg cell infiltration. Development of such inflammatory FOXP3lonon-Treg cells may depend on secretion of interleukin (IL)-12 and transforming growth factor (TGF)-β by tissues and their presence was correlated with tumor invasion by intestinal bacteria, especially Fusobacterium nucleatum. Thus, functionally distinct subpopulations of tumor-infiltrating FOXP3+ T cells contribute in opposing ways to determining CRC prognosis. Depletion of FOXP3hi Treg cells from tumor tissues, which would augment antitumor immunity, could thus be used as an effective treatment strategy for CRCs and other cancers, whereas strategies that locally increase the population of FOXP3lo non-Treg cells could be used to suppress or prevent tumor formation.