Patricia A. Masso-Welch

Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis

The commensal fungus Candida albicans causes oropharyngeal candidiasis (OPC; thrush) in settings of immunodeficiency. Although disseminated, vaginal, and oral candidiasis are all caused by C. albicans species, host defense against C. albicans varies by anatomical location. T helper 1 (Th1) cells have long been implicated in defense against candidiasis, whereas the role of Th17 cells remains controversial. IL-17 mediates inflammatory pathology in a gastric model of mucosal candidiasis, but is host protective in disseminated disease. Here, we directly compared Th1 and Th17 function in a model of OPC. Th17-deficient (IL-23p19−/−) and IL-17R–deficient (IL-17RA−/−) mice experienced severe OPC, whereas Th1-deficient (IL-12p35−/−) mice showed low fungal burdens and no overt disease. Neutrophil recruitment was impaired in IL-23p19−/− and IL-17RA−/−, but not IL-12−/−, mice, and TCR-αβ cells were more important than TCR-γδ cells. Surprisingly, mice deficient in the Th17 cytokine IL-22 were only mildly susceptible to OPC, indicating that IL-17 rather than IL-22 is vital in defense against oral candidiasis. Gene profiling of oral mucosal tissue showed strong induction of Th17 signature genes, including CXC chemokines and β defensin-3. Saliva from Th17-deficient, but not Th1-deficient, mice exhibited reduced candidacidal activity. Thus, the Th17 lineage, acting largely through IL-17, confers the dominant response to oral candidiasis through neutrophils and antimicrobial factors.

Prevention of Mammary Cancer with Conjugated Linoleic Acid: Role of the Stroma and the Epithelium

Conjugated linoleic acid (CLA), found naturally in dairy products and ruminant meats, refers to isomers of octadecadienoic acid with conjugated double bonds. CLA inhibits both DMBA- and NMU-induced rat mammary carcinogenesis, and its antitumor efficacy is similar whether it is fed only during puberty, or continuously during promotion. Pubertal feeding is associated with a reduced proliferation of the epithelial cells within the terminal end buds (TEBs) and lobular epithelium, and results in a decrease in the epithelial density, suggesting a reduction in the carcinogen-sensitive target population. During promotion, CLA feeding induces apoptosis of preneoplastic lesions. The effects of CLA are mediated by a direct action on the epithelium, as well as by an indirect effect through the stroma. CLA is incorporated into the neutral lipids of mammary adipocytes, where it can serve as a local reservoir of CLA. Additionally, CLA induces the adipogenic differentiation of multipotent mammary stromal cells in vitro, and inhibits their development into three-dimensional capillary networks. This suggested that CLA might inhibit angiogenesis in vivo, a hypothesis that was subsequently confirmed. The antiangiogenic effect is mediated, in part, through a CLA-induced decrease in serum VEGF (vascular endothelial growth factor) and mammary gland VEGF and flk-1. Together, the data suggest that CLA may be an excellent candidate for prevention of breast cancer.

A Developmental Atlas of Rat Mammary Gland Histology

The mammary gland is a dynamic tissue that undergoes epithelial expansion and invasion during puberty and cycles of branching and lobular morphogenesis, secretory differentiation, and regression during pregnancy, lactation, and involution. The alteration in the mammary gland epithelium during its postnatal differentiation is accompanied by changes in the multiple stromal cell types present in this complex tissue. The postnatal plasticity of the epithelium, endothelium, and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The purpose of this review is to assist researchers in recognizing histological changes in the epithelium and stroma of the rat mammary gland throughout development.

Altered expression and localization of PKC eta in human breast tumors

Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohisto chemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardlessof the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased inareas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.

SELECTIVE CHANGES IN EGF RECEPTOR EXPRESSION AND FUNCTION DURING THE PROLIFERATION, DIFFERENTIATION AND APOPTOSIS OF MAMMARY EPITHELIAL CELLS

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling.

Background: The mammary epithelium undergoes proliferation and regression accompanied by remodeling of the fibrocellular and vascular stroma. Mast cells are abundant in these compartments and have been implicated in remodeling during wound healing and cancer progression. The purpose of this study was to test the hypothesis that mast cell abundance correlates with physiologic mammary tissue remodeling during estrous cycling, lactogenesis (pregnancy and lactation) and involution. Results: Mast cell and capillary frequency were quantified in the stroma surrounding ducts and lobules from mammary glands of rats. During estrous cycling, periductal mast cell numbers were unchanged, but lobule‐associated mast cells significantly increased in the regressive phase of diestrus II. During lactogenesis, lobular stroma mast cells peaked early in pregnancy, at D2, followed by a significant decrease throughout lactation. Involution was associated with a rapid return in mast cell numbers, similar to diestrus II. Lobular vascularization peaked during the state of metestrus, when limited secretory differentiation occurs. Lobular angiogenesis peaked at D7 of pregnancy, regressed, and then returned to high levels during lactation and early involution, when secretory differentiation is high. Conclusions: These results suggest mast cells are predominantly associated with regressive lobular remodeling during cycling and involution, whereas angiogenesis is predominantly associated with secretory differentiation. Developmental Dynamics 241:890–900, 2012.

TLR2‐dependent modulation of dendritic cells by LT‐IIa‐B5, a novel mucosal adjuvant derived from a type II heat‐labile enterotoxin

A host of human pathogens invades the body at mucosal surfaces. Yet, strong, protective mucosal immune responses directed against those pathogens routinely cannot be induced without the use of adjuvants. Although the strongest mucosal adjuvants are members of the family of HLTs, the inherent toxicities of HLT holotoxins preclude their clinical use. Herein, it is shown that LT‐IIa‐B5 enhances mucosal immune responses by modulating activities of DCs. i.n. immunization of mice with OVA in the presence of LT‐IIa‐B5 recruited DCs to the NALT and significantly increased uptake of OVA by those DCs. Furthermore, LT‐IIa‐B5 increased expression of CCR7 by DCs, which mediated enhanced migration of the cells from the NALT to the draining CLNs. LT‐IIa‐B5 also enhanced maturation of DCs, as revealed by increased surface expression of CD40, CD80, and CD86. Ag‐specific CD4+ T cell proliferation was augmented in the CLNs of mice that had received i.n. LT‐IIa‐B5. Finally, when used as an i.n. adjuvant, LT‐IIa‐B5 dramatically increased the levels of OVA‐specific salivary IgA and OVA‐specific serum IgG. Strikingly, each of the activities induced by LT‐IIa‐B5 was strictly TLR2‐dependent. The data strongly suggest that the immunomodulatory properties of LT‐IIa‐B5 depend on the productive modulation of mucosal DCs. Notably, this is the first report for any HLT to demonstrate in vivo the elicitation of strong, TLR2‐dependent modulatory effects on DCs with respect to adjuvanticity.

Protein kinase C eta upregulation and secretion during postnatal rat mammary gland differentiation

The mammary gland has the ability to undergo repeated cycles of tightly regulated postnatal proliferation, differentiation, and apoptosis-mediated regression, providing a model to investigate potential regulators of mammary epithelial growth and differentiation. Protein kinase C eta is a candidate regulator of mammary epithelial differentiation, as increased expression of PKC eta is often observed during the terminal differentiation of many epithelial tissues. In this study, PKC eta expression and localization were characterized during puberty, pregnancy, lactation and involution in isolated rat mammary epithelial cells (MEC), as well as in paraffin-embedded and frozen rat mammary gland sections. By Western blot analysis of whole cell lysates from purified MEC, PKC eta protein expression increased during the shift from resting to a pregnant state. This increased PKC eta protein expression during pregnancy was associated with alveolar rather than ductal development, as immunohistochemical staining for PKC eta was increased in differentiating secretory alveoli, but not ducts. By immunofluorescent staining, PKC eta was stained intensely in an intracellular reticular meshwork throughout the cytosol of alveolar epithelial cells from pregnant mammary gland. During lactation, PKC eta was abundant in apocrine bodies budding from the alveolar epithelium, in the lumen of alveoli, and was present in milk, in association with casein, while being decreased in the cytoplasm of the luminal alveolar epithelium. Staining intensity of alveoli for PKC eta decreased further during involution. Western blotting of subcellular fractions from isolated mammary epithelial cells demonstrated that PKC eta remained associated with the membrane and particulate fractions throughout development. The upregulation of PKC eta in alveolar but not ductal epithelium during pregnancy suggests an association with functional secretory differentiation.

Chemopreventive and anti-angiogenic effects of dietary phenethyl isothiocyanate in an N-methyl nitrosourea-induced breast cancer animal model.

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen‐dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N‐methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor‐free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC‐treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC‐treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC‐treated animals. These studies indicate that supplementation with PEITC prolongs the tumor‐free survival, reduces tumor incidence and burden, and is chemoprotective in NMU‐induced estrogen‐dependent breast cancer in rats. For the first time, it is reported that PEITC has anti‐angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity. Copyright

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross‐linking treatment is a well‐described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr‐derived genes for ssDNA‐binding antibody also demonstrate a large percentage (35–75%) of immunoglobulin‐capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo. J. Leukoc. Biol. 66: 617–624; 1999.