Kyathanahalli S. Janardhan

Essential role of Orai1 store-operated calcium channels in lactation

Significance All mammals, from platypuses to humans, produce relatively immature offspring that are wholly dependent on their mother’s milk for their postnatal growth and development. However, the dynamic signaling and molecular mechanisms responsible for the transport of key constituents (e.g., calcium) into milk and for alveolar unit contraction and milk ejection are not fully understood. Using genetically modified mouse models, we demonstrate that the store-operated Ca2+ channel Orai1 delivers over 50% of the calcium ions present in milk. We also reveal an unanticipated role of Orai1 as a master regulator of oxytocin-mediated alveolar unit contractility, milk ejection, and pup survival. These results provide a unique mechanistic insight into the fundamentally mammalian process of lactation. The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca2+ channel subunit, Orai1, is required for both optimal Ca2+ transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca2+ in mammary myoepithelial cells followed by slow, irregular Ca2+ oscillations. These oscillations, and not the initial Ca2+ transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca2+ is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca2+ oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Obesity, rather than diet, drives epigenomic alterations in colonic epithelium resembling cancer progression.

While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.

Male infertility in mice lacking the store-operated Ca2+ channel Orai1

Store-operated calcium entry (SOCE) is an important Ca(2+) influx pathway in somatic cells. In addition to maintaining endoplasmic reticulum (ER) Ca(2+) stores, Ca(2+) entry through store-operated channels regulates essential signaling pathways in numerous cell types. Patients with mutations in the store-operated channel subunit ORAI1 exhibit defects in store-operated Ca(2+) influx, along with severe immunodeficiency, congenital myopathy and ectodermal dysplasia. However, little is known about the functional role of ORAI1 in germ cells and reproductive function in mice, or in men, since men with loss-of-function or null mutations in ORAI1 rarely survive to reproductive age. In this study, we investigated the role of ORAI1 in male reproductive function. We reveal that Orai1(-/-) male mice are sterile and have severe defects in spermatogenesis, with prominent deficiencies in mid- to late-stage elongating spermatid development. These studies establish an essential in vivo role for store-operated ORAI1 channels in male reproductive function and identify these channels as potential non-steroidal regulators of male fertility.

Key role for scavenger receptor B-I in the integrative physiology of host defense during bacterial pneumonia

Scavenger receptor B-I (SR-BI) is a multirecognition receptor that regulates cholesterol trafficking and cardiovascular inflammation. Although it is expressed by neutrophils (PMNs) and lung-resident cells, no role for SR-BI has been defined in pulmonary immunity. Herein, we report that, compared with SR-BI+/+ counterparts, SR-BI−/− mice suffer markedly increased mortality during bacterial pneumonia associated with higher bacterial burden in the lung and blood, deficient induction of the stress glucocorticoid corticosterone, higher serum cytokines, and increased organ injury. SR-BI−/− mice had significantly increased PMN recruitment and cytokine production in the infected airspace. This was associated with defective hematopoietic cell-dependent clearance of lipopolysaccharide from the airspace and increased cytokine production by SR-BI−/− macrophages. Corticosterone replacement normalized alveolar neutrophilia but not alveolar cytokines, bacterial burden, or mortality, suggesting that adrenal insufficiency derepresses PMN trafficking to the SR-BI−/− airway in a cytokine-independent manner. Despite enhanced alveolar neutrophilia, SR-BI−/− mice displayed impaired phagocytic killing. Bone marrow chimeras revealed this defect to be independent of the dyslipidemia and adrenal insufficiency of SR-BI−/− mice. During infection, SR-BI−/− PMNs displayed deficient oxidant production and CD11b externalization, and increased surface L-selectin, suggesting defective activation. Taken together, SR-BI coordinates several steps in the integrated neutrophilic host defense response to pneumonia.

Integrin β3 is not critical for neutrophil recruitment in a mouse model of pneumococcal pneumonia

Streptococcus pneumoniae is one of the most common causes of bacterial pneumonias in humans. Neutrophil migration into lungs infected with S. pneumoniae is central to the host defense but the mechanisms of neutrophil recruitment, as mediated by S. pneumoniae, into lungs are incompletely understood. Therefore, we have assessed the role of integrin αvβ3 by evaluating its subunit β3 in a mouse model of lung inflammation induced by S. pneumonia. Integrin subunit β3 knockout (β3-/-) and wild-type (WT) mice were intratracheally instilled with either S. pneumoniae or saline. Other groups of WT mice were treated intraperitoneally with 25xa0μg or 50xa0μg of antibody against integrin β3 or with isotype-matched antibody at 1xa0h before instillation of S. pneumoniae. Mice were killed 24xa0h after infection. Flow cytometry confirmed the absence or presence of integrin subunit β3 on peripheral blood neutrophils in β3-/- or WT mice, respectively. Neutrophil numbers in bronchoalveolar lavage (BAL) from infected β3-/- and WT mice showed no differences. Neutrophil numbers in BAL of infected WT mice treated with β3 antibody were lower compared with those without antibody but similar to those of mice administered isotype-matched antibody. Many neutrophils were present in the perivascular spaces of the lungs in β3-/- mice. Lungs from infected β3-/- mice had negligible mitogen-activated protein kinase expression compared with those of infected WT mice. Thus, integrin β3 or its heterodimer αvβ3 is not critical for neutrophil migration into lungs infected with S. pneumoniae.

Chapter 22 – Nose, Larynx, and Trachea

n Abstractn n Chapter 22 outlines the morphology of the nose, larynx, and trachea. The text includes descriptions of spontaneous and treatment-related lesions observed in the tissues in toxicity and carcinogenicity studies.n The text begins with a description of the normal upper respiratory tract through the stages of embryonic development. Anatomy and histology are discussed before delving into the various degenerative, regenerative, and adaptive lesions found in toxicology studies. Included in the text is information on inflammatory and vascular lesions, and how trauma and injury, as well as exposure to irritants, can elicit inflammatory responses.n Molecular pathology of the upper respiratory tract is investigated, as well as an examination of the molecular alterations within specific cell types, providing a better understanding of the mechanisms of tissue injury.n n

Histopathological and Immunohistochemical Characterization of Methyl Eugenol–induced Nonneoplastic and Neoplastic Neuroendocrine Cell Lesions in Glandular Stomach of Rats

Methyl eugenol induces neuroendocrine (NE) cell hyperplasia and tumors in F344/N rat stomach. Detailed histopathological and immunohistochemical (IHC) characterization of these tumors has not been previously reported. The objective of this study was to fill that data gap. Archived slides and paraffin blocks were retrieved from the National Toxicology Program Archives. NE hyperplasias and tumors were stained with chromogranin A, synaptophysin, amylase, gastrin, H+/K+ adenosine triphosphatase (ATPase), pepsinogen, somatostatin, and cytokeratin 18 (CK18) antibodies. Many of the rats had gastric mucosal atrophy, due to loss of chief and parietal cells. The hyperplasias and tumors were confined to fundic stomach, and females were more affected than the males. Hyperplasia of NE cells was not observed in the pyloric region. Approximately one-third of the females with malignant NE tumors had areas of pancreatic acinar differentiation. The rate of metastasis was 21%, with liver being the most common site of metastasis. Immunohistochemically, the hyperplasias and tumors stained consistently with chromogranin A and synaptophysin. Neoplastic cells were also positive for amylase and CK18 and negative for gastrin, somatostatin, H+/K+ ATPase, and pepsinogen. Metastatic neoplasms histologically similar to the primary neoplasm stained positively for chromogranin A and synaptophysin. Based on the histopathological and IHC features, the neoplasms appear to arise from enterochromaffin-like cells.

Immunohistochemistry in Investigative and Toxicologic Pathology

Immunohistochemistry (IHC) is a valuable tool in pathology. This review provides a brief description of the technical aspects of IHC and a detailed discussion on the variables that affect the results, interpretation, and reproducibility of IHC results. Lists of antibodies that have and have not worked in IHC on various mouse and rat tissues in our laboratory are provided as a guidance for selection of antibodies. An approach to IHC method optimization is presented. Finally, the critical information that should be included as a part of peer-reviewed manuscript is also discussed.

Chapter 23 – Lung, Pleura, and Mediastinum

n Abstractn n The lung is constantly exposed to a large volume of inhaled air that may contain toxicant xenobiotics. With the possibility of exposure to a variety of respiratory toxicants from airborne pollutants in our environment during the course of daily activities, in occupational settings, the use of aerosol sprays for household products, and the development of inhalant bronchial therapies, pulmonary toxicology has become an important subspecialty of toxicology. The lung is susceptible to injury following hematogenous exposure to toxicants. Susceptibility to injury and the type of response following exposure to air- or blood-borne toxicants is largely dependent on the physiochemical characteristics and concentration of the toxicant, duration of exposure, site/tissue specific sensitivity, and the integrity of the defense mechanisms of the lung. In this chapter, nonneoplastic and neoplastic spontaneous lesions and those that develop in the lungs of rats following exposure to toxicants by various routes, but primarily by inhalation, are discussed in detail which provides insight into our understanding of how human lungs respond to toxic chemicals. In addition, the gross and microscopic anatomy of the rat lung is also discussed some detail. Although inhalation is the primary route of exposure in experimental studies, in the past, many studies used intratracheal instillation or direct injection of known carcinogens into the lung. These experiments often resulted in the development of squamous cell carcinomas even though they are very rare as a naturally occurring neoplasm. Instillation of chemicals or particles into the trachea or pleura or direct injection into the lung results in lesions or responses that may not be as relevant to understanding the mechanism of pulmonary carcinogenesis as inhalation of materials under more normal conditions. There remain, however, many areas where our understanding of the response of the lung to toxic chemicals is incomplete.n n

NTP/NIEHS Global Contributions to Toxicologic Pathology

National Toxicology Program (NTP) pathologists are engaged in important initiatives that have significant global impact. These initiatives build on its leadership in pathology peer review and publications in the areas of toxicologic pathology, clinical pathology, and laboratory animal medicine. Over the past decade, NTP/National Institute of Environmental Health Sciences research initiatives have focused on cancer and noncancer hazard identification, with the goal of understanding cellular and molecular mechanisms of disease. New initiatives of significant global impact include the web-based nonneoplastic lesion atlas and an NTP partnership with international scientists to investigate molecular mechanisms at the whole genome level, which will be used to inform potential mechanisms of environmental exposures in human cancers. Also, we are dedicated to contributing to pathology and toxicology organizations through service on executive committees and editorial boards, participating in international projects and symposiums, and providing training for future leaders in toxicologic pathology. Herein, we provide highlights of our global contributions.