Pulmonary Granulomas Research Articles

Using Eggs from <em>Schistosoma mansoni</em> as an <em>In vivo</em> Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Imunofenotipagem e remodelamento da matriz extracelular na sarcoidose pulmonar e extrapulmonar

To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Fibrocytes contribute to the increased levels of fibrosis observed in Th2 pulmonary granulomas in TLR9-/- mice (54.18)

Abstract Despite many recent advances in the field, the underlying mechanisms governing pulmonary fibrosis remain poorly understood. We have used an experimental system of fibrotic granuloma development via the embolization of Schistosoma mansoni eggs to the lungs. Egg embolization results in the release of highly antigenic glycoproteins (Schistosoma egg antigen,SEA) that promote a dominant Th2 immune response. We previously demonstrated that in comparison to wild-type mice, TLR9-/- mice showed increased granuloma size and augmented collagen deposition. In the present study, we investigated the mechanisms underlying the fibrotic change of this Th2-driven pulmonary granuloma system, and the contribution of TLR9 signals to this phenomenon. The chemokine receptor/ligand pair CXCR4/CXCL12 has been shown to play an important role in the homing of fibrocytes; these cells express CXCR4 and migrate in response to CXCL12 in a model of bleomycin-induced pulmonary fibrosis. Levels of CXCL12 were higher in the lungs of mice treated with S. mansoni eggs, but there were no difference between wild-type and TLR9-/- mice. Flow cytometry data indicated that TLR9-/- mice showed increased number of CD45+Col I+CXCR4+ fibrocytes in the lungs of TLR9-/- mice treated with S. mansoni eggs in comparison to wild-type mice. Thus, our results suggested that increased fibrocytes may contribute to the increased levels of fibrosis observed in TLR9-/- mice.

Pulmonary Langerhans histiocytosis and eosinophilic granuloma.

A forty-tree-year-old female patient presented at the emergency department for low back pain and left hip pain since four weeks, mostly at night, and disabled walking. Neurological examination and laboratory findings were normal. Computed tomography of the lumbar spine showed degenerative discopathy at level L4-L5 and L5-S1, but revealed no other lesions explaining the patient’s acute complaints. Additional plain radiograph of the left hip revealed an oval cyst-like lucent lesion in the left iliopubic ramus with thinning of the adjacent cortical margin (Fig. A). A magnetic resonance imaging (MRI) for further exploration was proposed. The MRI revealed a T1 hypointense lesion surrounded by increased signal intensity on T2 images, consistent to an osteolytic lesion with peri-lesional bone marrow edema, and a concomitant fracture of the iliopubic ramus. SPECTCT confirmed the presence of a lytic bone lesion in the left iliopubic ramus with high tracer intensity (Fig. B), as well as three smaller osteolytic lesions in both ileum bones without tracer uptake. Considering the possibility of bone metastasis, thoracic and abdominal CT examination was performed to rule out a primary tumor. Both lungs showed multiple small cysts with irregular wall thickness, as well as numerous millimetric pulmonary nodules with groundglass and more dense appearance, both localized in the upper and middle lung zones, with sparing of the lung bases and costophrenic angles (Fig. C). The diagnosis of pulmonary Langerhans cell histiocytosis and eosinophilic granuloma was suggested. An ultrasound-guided percutaneous biopsy of the bone lesion in the left iliopubic ramus was performed. Histological and immunohistochemical examination of the biopt revealed a cluster of eosinophilic granulocyts staining for S100 protein and CD1 antigen, a specific Langerhans cell marker. The diagnosis of ‘pulmonary Langerhans cell histiocytosis’ and ‘eosinophilic granuloma’ was confirmed.

Pulmonary Hyalinizing Granuloma With Associated Elevation in Serum and Tissue IgG4 Occurring in a Patient With a History of Sarcoidosis

Pulmonary hyalinizing granulomas (PHGs) are unusual fibrosclerotic inflammatory lung lesions. The organ-based manifestations of the recently defined IgG4-related sclerosing disease typically show dense fibrosis and heavy lymphoplasmacytic infiltrates. IgG4-related sclerosing disease is also defined by increased serum IgG4 levels and increased tissue levels of IgG4-positive plasma cells. The morphologic features of PHG overlap with those seen in IgG4-related sclerosing disease, and this suggests that PHG may be a form of IgG4-related sclerosing disease. We present a case of a 51-year-old man with a history of sarcoidosis who presented with slowly enlarging pulmonary nodules. Histologic evaluation of one of the nodules yielded a diagnosis of PHG. Further investigation demonstrated both elevated serum IgG4 and elevated tissue IgG4-positive plasma cells in the PHG. In previous reports, lesions that are now considered part of IgG4-related sclerosing disease were documented in patients also diagnosed with PHG, although these reports date from before the description of IgG4 sclerosing disease. This case provides the first definitive evidence that PHG is part of the spectrum of IgG4-related sclerosing disease.

Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence.

Trehalose 6, 6’dimycolate (cord factor) induced pulmonary granuloma in naive and lymphopenic mice

Trehalose 6, 6’dimycolate (cord factor) induced pulmonary granuloma in naive and lymphopenic mice

Persistent Inflammation in Pulmonary Granuloma 48 Years after Talcage Pleurodesis, Detected by FDG-PET/CT

In patients with suspicion of lung malignancy, FDG PET/CT is frequently used as a diagnostic and staging imaging modality. However, false positive findings are not uncommon. We demonstrate a case with FDG-avid pulmonary nodules, mimicking lung cancer. After histopathological examination they appeared to be the result of persistent inflamed tissue, due to talcage pleurodesis, which occurred 48 years ago. We concluded that, nearly five decades after talcage pleurodesis, there can still be an ongoing inflammation reaction in the pleurae, which can be detected by FDG PET/CT.

A Short Interfering RNA (siRNA) Molecular Beacon for the Detection of Mycobacterial Infection

In latent TB, the ability of Mycobacterium tuberculosis to invade and survive within macrophages of the pulmonary granuloma is attributed to protein products of mammalian cell entry (mce4) operon genes (A-F). These are cholesterol transporters which facilitate the transport of host lipids into the mycobacterium allowing long term survival during chronic infection. Currently, there are no rapid and reliable tests for the detection of latent TB. Therefore, because there is a lack of reliable and efficient tests for the diagnosis of latent TB, we tested the hypothesis that mycobacterial infection can be detected using mce4 siRNA molecular beacons against mce4 mRNAs. Because our work showed that the mce4A gene of the mce4 operon conferred infectivity to host E. coli, a siRNA molecular beacon was designed against a region of the mce4A mRNA that is highly homologous in Mycobacterium tuberculosis and Mycobacterium smegmatis. This molecular beacon has a hairpin structure with a stem, 5 nucleotides on either end that are complementary to each other, and a loop which contains 20 nucleotides that are complementary to a region of the target mRNA. Conjugated to the 5’ and 3’ ends of the molecular beacon are the fluorophore TYE 665 and quencher Iowa Black RQ-SP. In the absence of the target mRNA the hairpin structure will predominate and fluorescence will be quenched while in the presence of the target mRNA fluorescence will be induced. Our study shows that the siRNA molecular beacon detects its target in M. smegmatis and in macrophages infected with M. smegmatis and offers a potential test for detection of mycobacterial infection.

Tolerance and Inflammation at the Gut Mucosa

Tolerance and Inflammation at the Gut Mucosa

BCG Induces Protection against Mycobacterium tuberculosis Infection in the Wistar Rat Model

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4+ and CD8+ T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8+ cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.

Computed Tomography of Granulomatous Pneumonia with Oxalosis in an American Alligator (Alligator mississippiensis) Associated withMetarhizium anisopliaevaranisopliae

An 18-yr-old, male, albino, American alligator (Alligator mississippiensis) was evaluated for decreased appetite and abnormal buoyancy. Computed tomography (CT) of the coelomic cavity showed multifocal mineral and soft tissue attenuating pulmonary masses consistent with pulmonary fungal granulomas. Additionally, multifocal areas of generalized, severe emphysema and pulmonary and pleural thickening were identified. The alligator was euthanized and necropsy revealed severe fungal pneumonia associated with oxalosis. Metarhizium anisopliae var. anisopliae was cultured from lung tissue and exhibited oxalate crystal formation in vitro. Crystals were identified as calcium oxalate monohydrate by X-ray powder defractometry. Fungal identification was based on morphology, including tissue sporulation, and DNA sequence analysis. This organism is typically thought of as an entomopathogen. Clinical signs of fungal pneumonia in nonavian reptiles are often inapparent until the disease is at an advanced stage, making antemortem diagnosis challenging. This case demonstrates the value of CT for pulmonary assessment and diagnosis of fungal pneumonia in the American alligator. Fungal infection with associated oxalosis should not be presumed to be aspergillosis.

Causes of pulmonary granulomas: a retrospective study of 500 cases from seven countries

BackgroundThe frequencies of various causes of pulmonary granulomas in pathological material are unknown, as is the influence of geographical location on aetiology. The aim of this study was to identify...

Indirect Effects of Oral Tolerance Inhibit Pulmonary Granulomas toSchistosoma mansoniEggs

Parenteral injection of tolerated proteins into orally tolerant mice inhibits the initiation of immunological responses to unrelated proteins and blocks severe chronic inflammatory reactions of immunological origin, such as autoimmune reactions. This inhibitory effect which we have called “indirect effects of oral tolerance” is also known as “bystander suppression.” Herein, we show that i.p. injection of OVA + Al(OH)3 minutes before i.v. injection of Schistosoma mansoni eggs into OVA tolerant mice blocked the increase of pulmonary granulomas. In addition, the expression of ICAM-1 in lung parenchyma in areas outside the granulomas of OVA-orally tolerant mice was significantly reduced. However, at day 18 after granuloma induction there was no difference in immunofluorescency intensity to CD3, CD4, F4/80, andα-SMA per granuloma area of tolerant and control groups. Reduction of granulomas by reexposure to orally tolerated proteins was not correlated with a shift in Th-1/Th-2 cytokines in serum or lung tissue extract.

Fatal Subarachnoid Hemorrhage Due to Ruptured Vertebrobasilar Aneurysm Associated With Giant Cell Arteritis in a Child

A 3 ½-year-old previously healthy female experienced an episode of sudden unresponsiveness witnessed by her mother. Upon arrival to the local hospital, imaging studies of the still unresponsive child revealed severe bilateral "flash" pulmonary edema and diffuse anoxic brain injury. Aggressive resuscitative efforts were unsuccessful, and she was pronounced dead. External examination at autopsy was essentially unremarkable. Internal examination of the head revealed diffuse basilar subarachnoid blood originating from a collapsed, 2 cm irregular aneurysm arising from the junction of the vertebral and basilar arteries. Additionally, multiple calcified subpleural, parenchymal, and hilar nodal pulmonary granulomas were identified. The remaining internal examination, including that of the aorta and its major branches, was unremarkable. Histologic examination of the aneurysm revealed alternating mural attenuation and thickening, the latter resulting from prominent intimal proliferation with active fibroplasia. Most notably, numerous isolated and clustered multinucleated giant cells were seen, most prominently in areas of more intense inflammation. Specific immunolabeling and silver staining of the pulmonary granulomas revealed evidence of histoplasmosis, but both were negative for fungal elements in the aneurysm, as was ultrastructural examination. The cause of death is fatal subarachnoid hemorrhage due to rupture of a vertebrobasilar artery aneurysm caused by isolated intracranial giant cell arteritis.

Stereological analysis of bacterial load and lung lesions in nonhuman primates (rhesus macaques) experimentally infected with Mycobacterium tuberculosis

Infection with Mycobacterium tuberculosis primarily produces a multifocal distribution of pulmonary granulomas in which the pathogen resides. Accordingly, quantitative assessment of the bacterial load and pathology is a substantial challenge in tuberculosis. Such assessments are critical for studies of the pathogenesis and for the development of vaccines and drugs in animal models of experimental M. tuberculosis infection. Stereology enables unbiased quantitation of three-dimensional objects from two-dimensional sections and thus is suited to quantify histological lesions. We have developed a protocol for stereological analysis of the lung in rhesus macaques inoculated with a pathogenic clinical strain of M. tuberculosis (Erdman strain). These animals exhibit a pattern of infection and tuberculosis similar to that of naturally infected humans. Conditions were optimized for collecting lung samples in a nonbiased, random manner. Bacterial load in these samples was assessed by a standard plating assay, and granulomas were graded and enumerated microscopically. Stereological analysis provided quantitative data that supported a significant correlation between bacterial load and lung granulomas. Thus this stereological approach enables a quantitative, statistically valid analysis of the impact of M. tuberculosis infection in the lung and will serve as an essential tool for objectively comparing the efficacy of drugs and vaccines.

Leptospirosis and Tularaemia in Raccoons (Procyon lotor) of Larimer Country, Colorado

SummaryRaccoons (Procyon lotor) are commonly implicated as carriers of many zoonotic pathogens. The purpose of this cross‐sectional study was to look for Leptospira interrogans and Francisella tularensis in opportunistically sampled, free‐ranging raccoons of Larimer Country, Colorado, USA. Sixty‐five animals were included in the study and testing consisted of gross post‐mortem examination, histopathology, and both immunohistochemistry and PCR for L. interrogans and F. tularensis. No significant gross lesions were identified and the most common histological lesions were lymphoplasmacytic interstitial nephritis and pulmonary silicosis; rare periportal hepatitis, splenic lymphoid hyperplasia and small pulmonary granulomas were also identified. Of 65 animals, 20 (30%) were positive for Leptospira on IHC but only one by PCR. Animals with inflammation in their kidneys were seven times more likely to be positive for Leptospira than animals without inflammation. The severity of inflammation was variable but often mild with minimal associated renal pathology. One animal was positive for Francisella on both IHC and PCR; IHC staining was localized to histiocytic cells within a pulmonary granuloma. In Colorado the significance and epidemiology of Leptospira is poorly understood. The high prevalence of infection in raccoons in this study population suggests that this species may be important in the regional epidemiology or could be used to estimate risk to domestic animals and humans. Identification of a single Francisella positive animal is significant as this is an uncommon disease in terrestrial animals within the state; the apparently higher prevalence in this peridomestic species implies that raccoons may be good indicators of the pathogen in the region. The results of this study suggest that raccoons may serve as effective sentinels for both Leptospira and Francisella in the state of Colorado. Further studies are needed to better characterize the prevalence and epidemiology of both organisms within the region.

Augmented Th2 response and liver fibrosis in mice lacking the chitinase like protein, BRP-39 (58.9)

Abstract The mouse Breast Regression Protein 39 (BRP39; Chi3l1) is a chitinase like protein but lacks enzymatic activity. Recently it was shown that mice lacking BRP39 displayed significantly impaired Th2 response during allergic airway disease. We sought to examine if the absence of BRP39 would affect the generation of protective and pathogenic Th2 responses following helminth infections. In a model of Schistosoma mansoni egg induced pulmonary granuloma formation, naive BRP39KO animals developed weaker Th2 response upon egg embolization. The Th2 responses were restored to WT levels if pulmonary granulomas were elicited in antigen primed (secondary granuloma) animals. However, the size of secondary egg granulomas remained significantly smaller in the KO animals, suggesting a role for BRP39 in regulation of pulmonary granuloma response. In contrast, following infection with S. mansoni cercariae, BRP39KO animals manifested larger hepatic granulomas and significantly exacerbated liver fibrosis and showed an increased prevalence of CD4+ cells making IL-5 and IL-13 among the granuloma leukocytes. Furthermore, Nippostrongylus brasiliensis infected BRP39KO mice displayed no defect in worm expulsion suggesting normal Th2 response in the gut. Thus, these studies reveal that the defectiveTh2 response in the absence of BRP39 is a transient phenomenon and introduce the possibility that BRP39 could negatively regulate Th2 responses in a chronic setting and in a tissue specific manner.

Pulmonary Mycobacterial Granuloma: Increased IL-10 Production Contributes to Establishing a Symbiotic Host–Microbe Microenvironment

The granuloma, a hallmark of host defense against pulmonary mycobacterial infection, has long been believed to be an active type 1 immune environment. However, the mechanisms regarding why granuloma fails to eliminate mycobacteria even in immune-competent hosts, have remained largely unclear. By using a model of pulmonary Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, we have addressed this issue by comparing the immune responses within the airway luminal and granuloma compartments. We found that despite having a similar immune cellular profile to that in the airway lumen, the granuloma displayed severely suppressed type 1 immune cytokine but enhanced chemokine responses. Both antigen-presenting cells (APCs) and T cells in granuloma produced fewer type 1 immune molecules including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide. As a result, the granuloma APCs developed a reduced capacity to phagocytose mycobacteria and to induce T-cell proliferation. To examine the molecular mechanisms, we compared the levels of immune suppressive cytokine IL-10 in the airway lumen and granuloma and found that both granuloma APCs and T cells produced much more IL-10. Thus, IL-10 deficiency restored type 1 immune activation within the granuloma while having a minimal effect within the airway lumen. Hence, our study provides the first experimental evidence that, contrary to the conventional belief, the BCG-induced lung granuloma represents a symbiotic host-microbe microenvironment characterized by suppressed type 1 immune activation.

Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.