Jose-Andres C. Portillo

CD40 Signaling in Macrophages Induces Activity against an Intracellular Pathogen Independently of Gamma Interferon and Reactive Nitrogen Intermediates

ABSTRACT Gamma interferon (IFN-γ) is the major inducer of classical activation of macrophages. Classically activated mouse macrophages acquire antimicrobial activity that is largely dependent on the production of reactive nitrogen intermediates. However, protection against important intracellular pathogens can take place in the absence of IFN-γ and nitric oxide synthase 2 (NOS2). Using Toxoplasma gondii as a model, we investigated if CD40 signaling generates mouse macrophages with effector function against an intracellular pathogen despite the absence of priming with IFN-γ and lack of production of reactive nitrogen intermediates. CD40-stimulated macrophages acquired anti-T. gondii activity that was not inhibited by a neutralizing anti-IFN-γ monoclonal antibody but was ablated by the neutralization of tumor necrosis factor alpha (TNF-α). Moreover, while the induction of anti-T. gondii activity in response to CD40 stimulation was unimpaired in macrophages from IFN-γ−/− mice, macrophages from TNF receptor 1/2−/− mice failed to respond to CD40 engagement. In contrast to IFN-γ-lipopolysaccharide, CD40 stimulation did not induce NOS2 expression and did not trigger production of reactive nitrogen intermediates. Neither NG-monomethyl-l-arginine nor diphenyleneiodonium chloride affected the induction of anti-T. gondii activity in response to CD40. Finally, macrophages from NOS2−/− mice acquired anti-T. gondii activity in response to CD40 stimulation that was similar to that of macrophages from wild-type mice. These results demonstrate that CD40 induces the antimicrobial activity of macrophages against an intracellular pathogen despite the lack of two central features of classically activated macrophages: priming with IFN-γ and production of reactive nitrogen intermediates.

CD40 Induces Anti-Toxoplasma gondii Activity in Nonhematopoietic Cells Dependent on Autophagy Proteins

ABSTRACT Toxoplasma gondii infects both hematopoietic and nonhematopoietic cells and can cause cerebral and ocular toxoplasmosis, as a result of either congenital or postnatally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is a key factor for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti-T. gondii activity at the level of nonhematopoietic cells. Engagement of CD40 on various endothelial cells including human microvascular brain endothelial cells, human umbilical vein endothelial cells, and a mouse endothelial cell line as well as human and mouse retinal pigment epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux, and led to recruitment of LC3 around the parasite. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40-stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various nonhematopoietic cells dependent on proteins of the autophagy machinery.

CD40 and tumour necrosis factor-α co-operate to up-regulate inducuble nitric oxide synthase expression in macrophages.

Regulation of inducible nitric oxide synthase (NOS2) expression is important given the role of this enzyme in inflammation, control of infections and immune regulation. In contrast to tumour necrosis factor‐α (TNF‐α) alone or CD40 stimulation alone, simultaneous stimulation of mouse macrophages through CD40 ligation and TNF‐α led to up‐regulation of NOS2 and nitric oxide production. This response was of functional relevance because CD40/TNF‐α‐stimulated macrophages acquired nitric oxide‐dependent anti‐Leishmania major activity. CD40 plus TNF‐α up‐regulated NOS2 independently of interferon‐γ, interferon‐α/β and interleukin‐1. TNF receptor‐associated factor 6 (TRAF6), an adapter protein downstream of CD40, appears to be required for NOS2 up‐regulation because a CD40‐TRAF6 blocking peptide inhibited up‐regulation of NOS2 in CD40/TNF‐α‐stimulated macrophages. CCAAT/enhancer‐binding protein‐β (C/EBPβ), a transcription factor activated by TNF‐α but not CD40, was required for NOS2 up‐regulation because this enzyme was not up‐regulated when C/EBPβ−/− macrophages received CD40 plus TNF‐α stimulation. These results indicate that CD40 and TNF‐α co‐operate to up‐regulate NOS2, probably via the effect of TRAF6 and C/EBPβ.

Blockade of CD40-TRAF2,3 or CD40-TRAF6 is sufficient to inhibit pro-inflammatory responses in non-haematopoietic cells.

Inhibition of the CD40–CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti‐CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40‐driven inflammatory disorders. Blocking tumour necrosis factor receptor‐associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II+ cells diminishes inflammation. However, CD40–TRAF6 blockade may cause immunosuppression. We examined the role of CD40–TRAF2,3 and CD40–TRAF6 signalling in the development of pro‐inflammatory responses in human non‐haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild‐type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non‐haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154‐induced up‐regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM‐1), monocyte chemotactic protein 1 (MCP‐1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM‐1 up‐regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP‐1 and tissue factor up‐regulation in these cells. Treatment of endothelial and smooth muscle cells with cell‐permeable peptides that block CD40–TRAF2,3 or CD40–TRAF6 signalling impaired pro‐inflammatory responses. In contrast, while the CD40–TRAF2,3 blocking peptide did not reduce CD154‐induced dendritic cell maturation, the CD40–TRAF6 blocking peptide impaired this response. Hence, preventing CD40–TRAF2,3 or CD40–TRAF6 interaction inhibits pro‐inflammatory responses in human non‐haematopoietic cells. In contrast to inhibition of CD40–TRAF6 signalling, inhibition of CD40–TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40–TRAF2,3 signalling may control CD40–CD154‐dependent inflammatory disorders.

Identification of Signaling Pathways by Which CD40 Stimulates Autophagy and Antimicrobial Activity against Toxoplasma gondii in Macrophages.

ABSTRACT CD40 is an important stimulator of autophagy and autophagic killing of Toxoplasma gondii in host cells. In contrast to autophagy induced by nutrient deprivation or pattern recognition receptors, less is known about the effects of cell-mediated immunity on Beclin 1 and ULK1, key regulators of autophagy. Here we studied the molecular mechanisms by which CD40 stimulates autophagy in macrophages. CD40 ligation caused biphasic Jun N-terminal protein kinase (JNK) phosphorylation. The second phase of JNK phosphorylation was dependent on autocrine production of tumor necrosis factor alpha (TNF-α). TNF-α and JNK signaling were required for the CD40-induced increase in autophagy. JNK signaling downstream of CD40 caused Ser-87 phosphorylation of Bcl-2 and dissociation between Bcl-2 and Beclin 1, an event known to stimulate the autophagic function of Beclin 1. However, TNF-α alone was unable to stimulate autophagy. CD40 also stimulated autophagy via a pathway that included calcium/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and ULK1. CD40 caused AMPK phosphorylation at its activating site, Thr-172. This effect was mediated by CaMKKβ and was not impaired by neutralization of TNF-α. CD40 triggered AMPK-dependent Ser-555 phosphorylation of ULK1. CaMKKβ, AMPK, and ULK1 were required for CD40-induced increase in autophagy. CD40-mediated autophagic killing of Toxoplasma gondii is known to require TNF-α. Knockdown of JNK, CaMKKβ, AMPK, or ULK1 prevented T. gondii killing in CD40-activated macrophages. The second phase of JNK phosphorylation—Bcl-2 phosphorylation—Bcl-2–Beclin 1 dissociation and AMPK phosphorylation-ULK1 phosphorylation occurred simultaneously at ∼4 h post-CD40 stimulation. Thus, CaMKKβ and TNF-α are upstream molecules by which CD40 acts on ULK1 and Beclin 1 to stimulate autophagy and killing of T. gondii.

CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

CD40, CX3CL1 and TNF-α promote atheroma and neointima formation. CD40 and TNF-α are also central to the development of diabetic retinopathy while CX3CL1 may play a role in the pathogenesis of this retinopathy. The purpose of this study was to examine whether CD40 ligation increases CX3CL1 and TNF-α protein expression in human endothelial cells from the aorta and retina. CD154 (CD40 ligand) upregulated membrane-bound and soluble CX3CL1 in human aortic endothelial cells. CD154 triggered TNF-α production by human aortic endothelial cells. TNF Receptor Associated Factors (TRAF) are key mediators of CD40 signaling. Compared to human aortic endothelial cells that express wt CD40, cells that express CD40 with a mutation that prevents TRAF2,3 recruitment, or CD40 with a mutation that prevents TRAF6 recruitment exhibited a profound inhibition of CD154-driven upregulation of membrane bound and soluble CX3CL1 as well as of TNF-α secretion. While both CD154 and TNF-α upregulated CX3CL1 in human aortic endothelial cells, these stimuli could act independently of each other. In contrast to human aortic endothelial cells, human retinal endothelial cells did not increase membrane bound or soluble CX3CL1 expression or secrete TNF-α in response to CD154 even though CD40 ligation upregulated ICAM-1 and CCL2 in these cells. Moreover, TNF-α did not upregulate CX3CL1 in retinal endothelial cells. In conclusion, CD40 ligation increases CX3CL1 protein levels and induces TNF-α production in endothelial cells. However, endothelial cells are heterogeneous in regards to these responses. Human aortic but not retinal endothelial cells upregulated CX3CL1 and TNF-α in response to CD40 ligation, as well as upregulated CX3CL1 in response to TNF-α. These dissimilarities may contribute to differences in regulation of inflammation in large vessels versus the retina.

Toxoplasma gondii induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy.

Targeting of Toxoplasma gondii by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, T. gondii activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.