Deanne Hebrink

P53 in nonneoplastic central nervous system lesions: An immunohistochemical and genetic sequencing study

OBJECTIVE Immunostaining for p53 commonly is considered a marker of neoplasia. Previous studies of nonneoplastic processes have yielded conflicting results. METHODS To test the assumption that p53 immunoreactivity indicates neoplasia, we examined 60 formalin-fixed, paraffin-embedded biopsies of nonneoplastic central nervous system lesions, including gliosis (n = 12), infarction (n = 9), demyelinating disease (n = 23), progressive multifocal leukoencephalopathy (n = 11), and herpes simplex virus encephalitis (n = 5). Diffuse astrocytomas (n = 50) of World Health Organization Grades 2 to 4 also were studied, as were six control autopsy brains . The avidin-biotin-peroxidase complex method was used with commercially available monoclonal antisera to both p53 (clone DO7; Dako, Carpinteria, CA) and mdm2 (Dako), a protein known to stabilize p53. Two samples of each nonneoplastic lesion also were subjected to deoxyribonucleic acid isolation, amplification, and sequencing of exons 5 to 8 of TP53. RESULTS Although it was low level in most instances, p53 immunoreactivity was noted in all but normal control samples. In reactive lesions, staining was largely observed in astrocytes and histiocytes. Scant oligodendroglia also were labeled in demyelinating disease. The progressive multifocal leukoencephalopathy samples revealed exceptionally strong staining in astrocytes and infected oligodendrocytes. Staining also was noted in occasional endothelial cells and neurons, and in rare lymphocytes. Immunoreactivity for mdm2, studied only in nonneoplastic lesions, was moderate to strong in all cases and limited to reactive astrocytes and histiocytes. No TP53 mutations were noted in the nonneoplastic lesions studied. To some extent, all astrocytomas exhibited p53 immunopositivity, particularly high-grade lesions. CONCLUSION p53 immunoreactivity is not limited to astrocytomas, but it can be observed in lesions that often are mistaken for glioma. No TP53 mutations accompany p53 expression in nonneoplastic lesions, and mdm2 may be responsible for persistence of p53 expression in these processes.

Glycosphingolipids Mediate Pneumocystis Cell Wall β-Glucan Activation of the IL-23/IL-17 Axis in Human Dendritic Cells

Pneumocystis species are opportunistic fungal organisms that cause severe pneumonia in immune-compromised hosts, with resultant high morbidity and mortality. Recent work indicates that IL-17 responses are important components of host defense against fungal pathogens. In the present study, we demonstrate that cell-surface β-glucan components of Pneumocystis (PCBG) stimulate human dendritic cells (DCs) to secrete IL-23 and IL-6. These cytokines are well established to stimulate a T helper-17 (Th17) phenotype. Accordingly, we further observe that PCBG-stimulated human DCs interact with lymphocytes to drive the secretion of IL-17 and IL-22, both Th17-produced cytokines. The activation of DCs was shown to involve the dectin-1 receptor with a downstream activation of the Syk kinase and subsequent translocation of both the canonical and noncanonical components of the NF-κB transcription factor family. Finally, we demonstrate that glycosphingolipid-rich microdomains of the plasma membrane participate in the activation of DCs by PCBG through the accumulation of lactosylceramide at the cell surface during stimulation with PCBG. These data strongly support the idea that the β-glucan surface components of Pneumocystis drive the activation of the IL-23/IL-17 axis during this infection, through a glycosphingolipid-initiated mechanism.

Evidence for Proinflammatory β-1,6 Glucans in the Pneumocystis carinii Cell Wall

ABSTRACT Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall β-glucans in stimulating inflammatory responses. Fungal β-glucans are comprised of d-glucose homopolymers containing β-1,3-linked glucose backbones with β-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized β-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for β-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize β-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific β-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) β-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized β-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate β-1,6 carbohydrate. In addition, isolated β-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-α) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that β-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection.

The Interaction of Pneumocystis with the C-Type Lectin Receptor Mincle Exerts a Significant Role in Host Defense against Infection

Pneumocystis pneumonia (PCP) remains a major cause of morbidity and mortality within immunocompromised patients. In this study, we examined the potential role of macrophage-inducible C-type lectin (Mincle) for host defense against Pneumocystis. Binding assays implementing soluble Mincle carbohydrate recognition domain fusion proteins demonstrated binding to intact Pneumocystis carinii as well as to organism homogenates, and they purified major surface glycoprotein/glycoprotein A derived from the organism. Additional experiments showed that rats with PCP expressed increased Mincle mRNA levels. Mouse macrophages overexpressing Mincle displayed increased binding to P. carinii life forms and enhanced protein tyrosine phosphorylation. The binding of P. carinii to Mincle resulted in activation of FcRγ-mediated cell signaling. RNA silencing of Mincle in mouse macrophages resulted in decreased activation of Syk kinase after P. carinii challenge, critical in downstream inflammatory signaling. Mincle-deficient CD4-depleted (Mincle−/−) mice showed a significant defect in organism clearance from the lungs with higher organism burdens and altered lung cytokine responses during Pneumocystis murina pneumonia. Interestingly, Mincle−/− mice did not demonstrate worsened survival during PCP compared with wild-type mice, despite the markedly increased organism burdens. This may be related to increased expression of anti-inflammatory factors such as IL-1Ra during infection in the Mincle−/− mice. Of note, the P. murina–infected Mincle−/− mice demonstrated increased expression of known C-type lectin receptors Dectin-1, Dectin-2, and MCL compared with infected wild-type mice. Taken together, these data support a significant role for Mincle in Pneumocystis modulating host defense during infection.

Differential macrophage polarization from Pneumocystis in immunocompetent and immunosuppressed hosts: potential adjunctive therapy during pneumonia

ABSTRACT We explored differential polarization of macrophages during infection using a rat model of Pneumocystis pneumonia. We observed enhanced pulmonary M1 macrophage polarization in immunosuppressed (IS) hosts, but an M2 predominant response in immunocompetent (IC) hosts following Pneumocystis carinii challenge. Increased inflammation and inducible nitric oxide synthase (iNOS) levels characterized the M1 response. However, macrophage ability to produce nitric oxide was defective. In contrast, the lungs of IC animals revealed a prominent M2 gene signature, and these macrophages effectively elicited an oxidative burst associated with clearance of Pneumocystis. In addition, during P. carinii infection the expression of Dectin-1, a critical receptor for recognition and clearance of P. carinii, was upregulated in macrophages of IC animals but suppressed in IS animals. In the absence of an appropriate cytokine milieu for M2 differentiation, Pneumocystis induced an M1 response both in vitro and in vivo. The M1 response induced by P. carinii was plastic in nature and reversible with appropriate cytokine stimuli. Finally, we tested whether macrophage polarization can be modulated in vivo and used to help manage the pathogenesis of Pneumocystis pneumonia by adoptive transfer. Treatment with both M1 and M2 cells significantly improved survival of P. carinii-infected IS hosts. However, M2 treatment provided the best outcomes with efficient clearance of P. carinii and reduced inflammation.

Dectin-2 Is a C-Type Lectin Receptor that Recognizes Pneumocystis and Participates in Innate Immune Responses

&NA; Pneumocystis is an important fungal pathogen that causes life‐threatening pneumonia in patients with AIDS and malignancy. Lung fungal pathogens are recognized by C‐type lectin receptors (CLRs), which bind specific ligands and stimulate innate immune responses. The CLR Dectin‐1 was previously shown to mediate immune responses to Pneumocystis spp. For this reason, we investigated a potential role for Dectin‐2. Rats with Pneumocystis pneumonia (PCP) exhibited elevated Dectin‐2 mRNA levels. Soluble Dectin‐2 carbohydrate‐recognition domain fusion protein showed binding to intact Pneumocystis carinii (Pc) and to native Pneumocystis major surface glycoprotein/glycoprotein A (Msg/gpA). RAW macrophage cells expressing V5‐tagged Dectin‐2 displayed enhanced binding to Pc and increased protein tyrosine phosphorylation. Furthermore, the binding of Pc to Dectin‐2 resulted in Fc receptor‐&ggr;‐mediated intracellular signaling. Alveolar macrophages from Dectin‐2‐deficient mice (Dectin‐2‐/‐) showed significant decreases in phospho‐Syk activation after challenge with Pc cell wall components. Stimulation of Dectin‐2‐/‐ alveolar macrophages with Pc components showed significant decreases in the proinflammatory cytokines IL‐6 and TNF‐&agr;. Finally, during infection with Pneumocystis murina, Dectin‐2‐/‐ mice displayed downregulated mRNA expression profiles of other CLRs implicated in fungal immunity. Although Dectin‐2‐/‐ alveolar macrophages had reduced proinflammatory cytokine release in vitro, Dectin‐2‐/‐ deficiency did not reduce the overall resistance of these mice in the PCP model, and organism burdens were statistically similar in the long‐term immunocompromised and short‐term immunocompetent PCP models. These results suggest that Dectin‐2 participates in the initial innate immune signaling response to Pneumocystis, but its deficiency does not impair resistance to the organism.

Binding of Pneumocystis carinii to the lung epithelial cell receptor HSPA5 (GRP78)

The importance of lung macrophages in Pneumocystis-host interaction is well known, but little is known about the initial binding/colonization of the airway epithelium. Our prior studies have documented cell-signalling events that occur following binding of the organisms to lung epithelial cells; however, the receptors that mediate Pneumocystis attachment to lung surfaces have not yet been fully defined. Using affinity chromatography, we identified heat shock protein 5 (HSPA5), also known as GRP78, as a potential host receptor that may have relevance in Pneumocystis lung colonization. Pneumocystis carinii (Pc) organisms not only bound HSPA5 on a rat lung epithelial cell line, but also on primary rat airway epithelial cells (AECs). Furthermore, Pc bound CHO1 cells overexpressing HSPA5 more than the CHO1 parent line alone, supporting a role for Pc-HSPA5 protein interaction in mediating organism attachment. These results provide new insights into the interactions of Pneumocystis with host lung epithelium.

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

&NA; N‐acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)‐GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP‐GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP‐GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc‐binding recombinant protein and a novel GlcNAc‐binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high‐performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP‐GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms.