Modeling of adaptive immunity uncovers disease tolerance mechanisms.

When an organism is challenged with a pathogen a cascade of events unfolds. The innate immune system rapidly mounts a preliminary nonspecific defense, while the acquired immune system slowly develops microbe-killing specialists. These responses cause inflammation, and along with the pathogen cause direct and indirect tissue damage, which anti-inflammatory mediators seek to temper. This interplay of systems is credited for maintaining homeostasis but may produce unexpected results such as disease tolerance. Tolerance is characterized by the persistence of pathogen and damage mitigation, where the relevant mechanisms are poorly understood. In this work we develop an ordinary differential equations model of the immune response to infection in order to identify key components in tolerance. Bifurcation analysis uncovers health, immune- and pathogen-mediated death clinical outcomes dependent on pathogen growth rate. We demonstrate that decreasing the inflammatory response to damage and increasing the strength of the immune system gives rise to a region in which limit cycles, or periodic solutions, are the only biological trajectories. We then describe areas of parameter space corresponding to disease tolerance by varying immune cell decay, pathogen removal, and lymphocyte proliferation rates.

Characterization of Immune Cells From the Lungs of Patients With Chronic Non-Tuberculous Mycobacteria or Pseudomonas aeruginosa Infection.

Little is known of the lung cellular immunophenotypes in patients with non-tuberculous mycobacterial lung disease (NTM-LD). Flow-cytometric analyses for the major myeloid and lymphoid cell subsets were performed in less- and more-diseased areas of surgically resected lungs from six patients with NTM-LD and two with Pseudomonas aeruginosa lung disease (PsA-LD). Lymphocytes, comprised mainly of NK cells, CD4+ and CD8+ T cells, and B cells, accounted for ~60% of all leukocytes, with greater prevalence of T and B cells in more-diseased areas. In contrast, fewer neutrophils were found with decreased number in more-diseased areas. Compared to NTM-LD, lung tissues from patients with PsA-LD demonstrated relatively lower numbers of T and B lymphocytes but similar numbers of NK cells. While this study demonstrated a large influx of lymphocytes into the lungs of patients with chronic NTM-LD, further analyses of their phenotypes are necessary to determine the significance of these findings.

Therapeutic efficacy of antimalarial drugs targeting DosRS signaling in Mycobacterium abscessus.

A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.

Mycobacterium avium Infection in a C3HeB/FeJ Mouse Model.

Infections caused by Mycobacterium avium complex (MAC) species are increasing worldwide, resulting in a serious public health problem. Patients with MAC lung disease face an arduous journey of a prolonged multidrug regimen that is often poorly tolerated and associated with relatively poor outcome. Identification of new animal models that demonstrate a similar pulmonary pathology as humans infected with MAC has the potential to significantly advance our understanding of nontuberculosis mycobacteria (NTM) pathogenesis as well as provide a tractable model for screening candidate compounds for therapy. One new mouse model is the C3HeB/FeJ which is similar to MAC patients in that these mice can form foci of necrosis in granulomas. In this study, we evaluated the ability of C3HeB/FeJ mice exposure to an aerosol infection of a rough strain of MAC 2285 to produce a progressive infection resulting in small necrotic foci during granuloma formation. C3HeB/FeJ mice were infected with MAC and demonstrated a progressive lung infection resulting in an increase in bacterial burden peaking around day 40, developed micronecrosis in granulomas and was associated with increased influx of CD4+ Th1, Th17, and Treg lymphocytes into the lungs. However, during chronic infection around day 50, the bacterial burden plateaued and was associated with the reduced influx of CD4+ Th1, Th17 cells, and increased numbers of Treg lymphocytes and necrotic foci during granuloma formation. These results suggest the C3HeB/FeJ MAC infection mouse model will be an important model to evaluate immune pathogenesis and compound efficacy.

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.

Iterative feature removal yields highly discriminative pathways.

BACKGROUND: We introduce Iterative Feature Removal (IFR) as an unbiased approach for selecting features with diagnostic capacity from large data sets. The algorithm is based on recently developed tools in machine learning that are driven by sparse feature selection goals. When applied to genomic data, our method is designed to identify genes that can provide deeper insight into complex interactions while remaining directly connected to diagnostic utility. We contrast this approach with the search for a minimal best set of discriminative genes, which can provide only an incomplete picture of the biological complexity. RESULTS: Microarray data sets typically contain far more features (genes) than samples. For this type of data, we demonstrate that there are many equivalently-predictive subsets of genes. We iteratively train a classifier using features identified via a sparse support vector machine. At each iteration, we remove all the features that were previously selected. We found that we could iterate many times before a sustained drop in accuracy occurs, with each iteration removing approximately 30 genes from consideration. The classification accuracy on test data remains essentially flat even as hundreds of top-genes are removed.Our method identifies sets of genes that are highly predictive, even when comprised of genes that individually are not. Through automated and manual analysis of the selected genes, we demonstrate that the selected features expose relevant pathways that other approaches would have missed. CONCLUSIONS: Our results challenge the paradigm of using feature selection techniques to design parsimonious classifiers from microarray and similar high-dimensional, small-sample-size data sets. The fact that there are many subsets of genes that work equally well to classify the data provides a strong counter-result to the notion that there is a small number of "top genes" that should be used to build classifiers. In our results, the best classifiers were formed using genes with limited univariate power, thus illustrating that deeper mining of features using multivariate techniques is important.

Low-cost aerosol exposure system for Guinea pigs.

BACKGROUND: This project designed and tested an economical apparatus to safely expose guinea pigs to biohazardous aerosol. The goals were to design a system that can be easily decontaminated, fits in a biosafety cabinet, and affordable. METHODS: It is composed of three main chambers housed in an outer box that fits within a conventionally sized biosafety cabinet. The animal chamber contains a removable housing unit for either four or eight guinea pigs. The aerosol chamber is separate to minimize contamination. The nebulizer chamber is also sealed to reduce risks from leakages. This apparatus is easily decontaminated by immersion in disinfectant. RESULTS AND CONCLUSIONS: This system has been tested for safety, ergonomics, efficiency of rodent exposure to bacteria, airflow, access points, seal mechanisms, and size. This system is effective, consistent, safe and cost efficient.

Lipoarabinomannan localization and abundance during growth of Mycobacterium smegmatis.

Lipoarabinomannan (LAM) is a structurally heterogeneous amphipathic lipoglycan present in Mycobacterium spp. and other actinomycetes, which constitutes a major component of the cell wall and exhibits a wide spectrum of immunomodulatory effects. Analysis of Mycobacterium smegmatis subcellular fractions and spheroplasts showed that LAM and lipomannan (LM) were primarily found in a cell wall-enriched subcellular fraction and correlated with the presence (or absence) of the mycolic acids in spheroplast preparations, suggesting that LAM and LM are primarily associated with the putative outer membrane of mycobacteria. During the course of these studies significant changes in the LAM/LM content of the cell wall were noted relative to the age of the culture. The LAM content of the M. smegmatis cell wall was dramatically reduced as the bacilli approached stationary phase, whereas LM, mycolic acid, and arabinogalactan content appeared to be unchanged. In addition, cell morphology and acid-fast staining characteristics showed variations with growth phase of the bacteria. In the logarithmic phase, the bacteria were found to be classic rod-shaped acid-fast bacilli, while in the stationary phase M. smegmatis lost the characteristic rod shape and developed a punctate acid-fast staining pattern with carbolfuchsin. The number of viable bacteria was independent of LAM content and phenotype. Taken together, the results presented here suggest that LAM is primarily localized with the mycolic acids in the cell wall and that the cellular concentration of LAM in M. smegmatis is selectively modulated with the growth phase.

Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis.

Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression profiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS.

Non-invasive diagnosis of early pulmonary disease in PECAM-deficient mice using infrared pulse oximetry.

Pulse oximetry is a common tool for detecting reduced pulmonary function in human interstitial lung diseases. It has not previously been used in a mouse model of interstitial lung disease. Further, platelet endothelial cell adhesion molecule deficient mice rarely show symptoms until disease is advanced. Using blood oxygen saturation, different stages of disease could be identified in a non-invasive manner. These stages could be correlated to pathology. Collagen deposition, using Picrosirius Red, did correlate with blood oxygen saturation. These studies are the first to show the use of an infrared pulse oximetry system to analyze the progression of a fibrotic interstitial lung disease in a mouse model of the human diseases. Further, these studies show that an early alveolar damage/enlargement event precedes the fibrosis in this mouse model, a stage that represents the best targets for disease analysis and prevention. This stage does not have extensive collagen deposition. Most importantly, targeting this earliest stage of disease for therapeutic intervention may lead to novel treatment for human disease.

Flexible low-cost system for small animal aerosol inhalation exposure to drugs, proteins, inflammatory agents, and infectious agents.

The design for a simple, low-cost aerosol generation system for rodent inhalation studies is described here. This system is appropriate for low biohazard-level agents. In this study, two biosafety level 2 agents, Pasturella pneumotropica and Pseudomonas aeruginosa, were tested successfully. This system was also used to immunize mice and guinea pigs in ovalbumin-based models of pulmonary inflammation. This design is appropriate for studies with limited budgets and lower-level biosafety containment.

PECAM-independent thioglycollate peritonitis is associated with a locus on murine chromosome 2.

BACKGROUND: Previous studies have demonstrated that knockout or inhibition of Platelet/Endothelial Cell Adhesion Molecule (PECAM, CD31) in a number of murine strains results in impaired inflammatory responses, but that no such phenotype is seen in the C57BL/6 (B6) murine background. METHODOLOGY/PRINCIPAL FINDINGS: We have undertaken a quantitative trait locus (QTL) mapping effort between FVB/n (FVB) and B6 mice deficient for PECAM to identify the gene or genes responsible for this unique feature of B6 mice. We have identified a locus on murine chromosome 2 at approximately 35.8 Mb that is strongly associated (LOD score = 9.0) with inflammatory responses in the absence of PECAM. CONCLUSIONS/SIGNIFICANCE: These data potentiate further study of the diapedesis machinery, as well as potential identification of new components of this machinery. As such, this study is an important step to better understanding the processes of inflammation.

Cloning and structural analysis of equine platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106).

Platelet endothelial cell adhesion molecule (PECAM, CD31) and vascular cell adhesion molecule-1 (VCAM-1, CD106) are essential for leukocyte emigration and diapedesis. PECAM is an essential histologic marker of endothelial cells; VCAM-1 is a prototype marker for endothelial cell activation. In this study, equine PECAM and VCAM mRNA were cloned and sequenced. Both genes are highly conserved amongst several species. This study also revealed conserved structural and regulatory motifs, emphasizing the importance of these genes' physiological roles in immunological responses.

The murine CD99-related molecule CD99-like 2 (CD99L2) is an adhesion molecule involved in the inflammatory response.

CD99, a glycoprotein found on the surfaces of leukocytes and concentrated at the borders of endothelial cells, plays a major role in the migration of leukocytes across endothelial cells into sites of inflammation, and has other roles in thymocyte development. The human and mouse genomes encode only two proteins related to CD99. One of these, XGA, is a red blood cell surface antigen. The function of the other, CD99-like 2 (CD99L2), is not known. We cloned mouse CD99L2 and used CD99L2 isolated from transfected cells to raise specific antibodies. Similar to human CD99, CD99L2 was expressed at the borders between transfected cells as well as on mouse leukocytes and vascular endothelial cells in situ. Transfection of L cell fibroblasts with CD99L2 imparted to them the ability to adhere to each other in a divalent cation-dependent, homophilic manner. Anti-CD99L2 antibody blocked influx of neutrophils and monocytes into a site of inflammation in vivo.

Different susceptibilities of PECAM-deficient mouse strains to spontaneous idiopathic pneumonitis.

Platelet Endothelial Cell Adhesion Molecule (PECAM) is an adhesion and signaling molecule used for leukocyte extravasation. We have generated two strains of PECAM-deficient mouse, one in the original C57BL/6 and a second by backcrossing nice generations into the FVB/n strain. The FVB/n strain has reduced responses in models of acute inflammation. We show here that this strain is also susceptible to a chronic pneumonia which leads to pulmonary fibrosis. In contrast, PECAM-deficient C57BL/6 mice do not develop this lung disease and have normal responses in acute models of inflammation. This demonstrates that PECAM-dependent and -independent mechanisms are found in both acute and chronic inflammation. Further, the PECAM-deficient FVB/n strain has many pathologic similarities to the human disease Idiopathic Pulmonary Fibrosis, suggesting that similar molecular mechanisms may play a role in human disease.

Platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains.

PECAM is a molecule used specifically during the diapedesis step when neutrophils and monocytes leave the blood compartment. Anti-PECAM reagents, such as Abs and soluble fusion proteins, block diapedesis both in vivo and in vitro. However, the PECAM knockout mouse in C57BL/6 strain has no serious defects in most models of inflammation. We show in this study that the same PECAM knockout backcrossed into the FVB/n strain clearly has reduced leukocyte emigration in two models of inflammation. Furthermore, we show that anti-PECAM reagents can block leukocyte emigration in several other wild-type strains of mice like FVB/n, SJL, and the outbred strain Swiss Webster. This clearly shows that the C57BL/6 strain is uniquely able to compensate for the loss of PECAM function. Murine models of inflammatory disease that have been studied using C57BL/6 mice should be re-evaluated using FVB/n or other mouse strains to determine whether PECAM plays a role in those models.

Locomotion of monocytes on endothelium is a critical step during extravasation.

Monocytes, like all leukocytes, undergo a series of sequential steps during extravasation from blood into tissues: tethering, rolling, adhesion and diapedesis. We have discovered an essential step, which we call locomotion, in which the monocyte moves from a site of firm adhesion to the nearest junction to begin diapedesis. Blocking CD11a-CD18 and CD11b-CD18 on human monocytes or adhesion molecules ICAM-1 and ICAM-2 on endothelial cells prevented the monocytes from reaching junctions. The blocked monocytes spun in circles as if they were unable to direct their movement despite being able to adhere and polarize normally. This step fills a gap in the paradigm of extravasation as a multistep process.

CD99 plays a major role in the migration of monocytes through endothelial junctions.

CD99 is a heavily O-glycosylated 32-kD type I transmembrane protein that is expressed on most hematopoietic cells. We show here that CD99 is expressed on endothelial cells and is concentrated at the borders between confluent cells. We found that a monoclonal antibody to CD99, hec2, selectively inhibited diapedesis of monocytes across endothelial cells by >90%. Diapedesis involved the homophilic interaction of CD99 on monocytes with CD99 on endothelial junctions. CD99 functioned distally to the point at which platelet-endothelial cell adhesion molecule 1 (PECAM-1, also known as CD31), another adhesion molecule involved in transmigration, played its critical role. Confocal microscopy showed that anti-PECAM-1 arrested leukocytes on the apical surface of endothelium, whereas blocking CD99 arrested monocytes at a point where they were partially through the junction. Therefore, diapedesis, the forward migration of leukocytes through endothelial junctions, is regulated sequentially by two distinct molecules, PECAM-1 and CD99.