No abstract

Toll-like receptor (TLR)9 performs our innate response to bacterial DNA, warning us of the presence of infection. Inhibitory oligodeoxyribonucleotides (INH-ODN) have been developed that selectively block activation of mouse TLR9. Their inhibitory motif consisting of CCx(not-C)(not-C)xxGGG (x = any base) also reduces anti-DNA antibodies in lupus mice. The current study demonstrates that this motif also provides the sequences required to block TLR9 in human B cells and human embryonic kidney (HEK) cells transfected with human TLR9. However, extending the sequence by four to five bases at the 5' end enhanced activity and this enhancement was greater when a phosphorothioate (pS) backbone replaced the native phosphodiester (pO) backbone. A series of pO-backbone INH-ODN representing a 500-fold range of activity in biologic assays was shown to cover less than a 2.5-fold range of avidity for binding human TLR9-Ig fusion protein, eliminating TLR9 ectodomain binding as the explanation for sequence-specific differences in biologic activity. With few exceptions, the relative activity of INH-ODN in Namalwa cells and HEK/human TLR9 cells was similar to that seen in mouse B cells. INH-ODN activity in human peripheral blood B cells correlated significantly with the cell line data. These results favor the conclusion that although the backbone determines strength of TLR9 binding, critical recognition of the INH-ODN sequence necessary for biologic activity is performed by a molecule that is not TLR9. These studies also identify the strongest INH-ODN for human B cells, helping to guide the selection of INH-ODN sequences for therapeutics in any situation where inflammation is enhanced by TLR9.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Allograft inflammatory factor 1 (AIF1) is a commonly used marker for microglia in the brains of humans and some animal models but has had limited applications elsewhere. We sought to determine whether AIF1 can be used as a macrophage marker across common laboratory animal species and tissues. We studied tissues (that is, spleen, liver, and lung) with defined macrophage populations by using an AIF1 immunostaining technique previously validated in human tissue. Tissues were collected from various mouse strains ( = 20), rat strains ( = 15), pigs ( = 4), ferrets ( = 4), and humans ( = 4, lung only). All samples of liver had scattered immunostaining in interstitial cells, consistent with resident tissue macrophages (Kupffer cells). Spleen samples had cellular immunostaining of macrophages in both the red and white pulp compartments, but the red pulp had more immunostained cellular aggregates and, in some species, increased immunostaining intensity compared with white pulp. In lung, alveolar macrophages had weak to moderate staining, whereas interstitial and perivascular macrophages demonstrated moderate to robust staining. Incidental lesions and tissue changes were detected in some sections, including a tumor, inducible bronchus-associated lymphoid tissue, and inflammatory lesions that demonstrated AIF1 immunostaining of macrophages. Finally, we compared AIF1 immunostaining of alveolar macrophages between a hypertensive rat model (SHR strain) and a normotensive model (WKY strain). SHR lungs had altered intensity and distribution of immunostaining in activated macrophages compared with macrophages of WKY lungs. Overall, AIF1 immunostaining demonstrated reproducible macrophage staining across multiple species and tissue types. Given the increasing breadth of model species used to study human disease, the use of cross-species markers and techniques can reduce some of the inherent variability within translational research.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.