Infected Apoptotic Cells Research Articles

RIP2 Contributes to Expanded CD4+ T Cell IFN-γ Production during Efferocytosis of Streptococcus pneumoniae–Infected Apoptotic Cells

Apoptotic cell clearance by professional and nonprofessional phagocytes in the process of efferocytosis is critical to preserve tissue homeostasis. Uptake of apoptotic cells by dendritic cells generates regulatory T cells and induces immunologic tolerance against self-antigens. In contrast, ingestion of infected apoptotic cells promotes activation of TLR4/MyD88-dependent bone marrow-derived dendritic cells (BMDCs) and triggers Th17 cell differentiation. In this study, we evaluated the impact of Streptococcus pneumoniae-infected apoptotic cell efferocytosis by BMDCs derived from C57BL/6 mice on differentiation and expansion of CD4+ T cell subsets, as well as the role of TLR2/4 and receptor-interacting protein 2 (RIP2) receptors in recognizing intracellular pathogens during efferocytosis. We demonstrated that BMDC-mediated efferocytosis of S. pneumoniae-infected apoptotic cells induced Th1 cell differentiation and expansion. Although TLR2/4 and RIP2 deficiency in BMDCs did not affect Th1 cell differentiation during efferocytosis, the absence of RIP2 decreased IFN-γ production by CD4 T cells during the expansion phase. These findings suggest that RIP2-mediated IL-1β production during efferocytosis of S. pneumoniae-infected apoptotic cells partially supports a Th1-mediated IFN-γ production microenvironment.

Distinctive role of efferocytosis in dendritic cell maturation and migration in sterile or infectious conditions.

Efferocytosis, or clearance of apoptotic cells (ACs), by dendritic cells (DCs) leads to immune response suppression and tolerance to self-antigens. However, efferocytosis of infected apoptotic cells (IACs) leads to the production of a mixed pro- and anti-inflammatory cytokine milieu. We examined the DC phenotype and ability to migrate after phagocytosis of ACs or IACs and observed higher levels of CD86 and CCR7 expression in DCs, as well as enhanced migration capacity following efferocytosis of IACs. Interestingly, higher levels of interleukin-1β, interleukin-10 and prostaglandin E2 (PGE2 ) were also produced in this context. Blockage of IAC recognition led to an impaired maturation profile and PGE2 production, which may have contributed to reduced CD86 and CCR7 expression and migration capacity. These data contribute to the understanding of how efferocytosis of sterile or infected cells may regulate the adaptive immune response, although the precise role of PGE2 in this process requires further investigation.

In vitro Antigen-presentation Assay for Self- and Microbial-derived Antigens.

Antigen presenting cells (APC) are able to process and present to T cells antigens from different origins. This mechanism is highly regulated, in particular by Patter Recognition Receptor (PRR) signals. Here, I detail a protocol designed to assess in vitro the capacity of APC to present antigens derived from bacteria, apoptotic and infected apoptotic cells.

Topical Prostaglandin E Analog Restores Defective Dendritic Cell-Mediated Th17 Host Defense Against Methicillin-Resistant Staphylococcus Aureus in the Skin of Diabetic Mice.

People with diabetes are more prone to Staphylococcus aureus skin infection than healthy individuals. Control of S. aureus infection depends on dendritic cell (DC)–induced T-helper 17 (Th17)–mediated neutrophil recruitment and bacterial clearance. DC ingestion of infected apoptotic cells (IACs) drive prostaglandin E2 (PGE2) secretion to generate Th17 cells. We speculated that hyperglycemia inhibits skin DC migration to the lymph nodes and impairs the Th17 differentiation that accounts for poor skin host defense in diabetic mice. Diabetic mice showed increased skin lesion size and bacterial load and decreased PGE2 secretion and Th17 cells compared with nondiabetic mice after methicillin-resistant S. aureus (MRSA) infection. Bone marrow–derived DCs (BMDCs) cultured in high glucose (25 mmol/L) exhibited decreased Ptges mRNA expression, PGE2 production, lower CCR7-dependent DC migration, and diminished maturation after recognition of MRSA-IACs than BMDCs cultured in low glucose (5 mmol/L). Similar events were observed in DCs from diabetic mice infected with MRSA. Topical treatment of diabetic mice with the PGE analog misoprostol improved host defense against MRSA skin infection by restoring DC migration to draining lymph nodes, Th17 differentiation, and increased antimicrobial peptide expression. These findings identify a novel mechanism involved in poor skin host defense in diabetes and propose a targeted strategy to restore skin host defense in diabetes.

‘Efferocytosis of infected apoptotic cells inhibits Th17 differentiation via PGE2-EP4 signaling’

Abstract Clearance of infected apoptotic cells (IAC) by dendritic cells (DC) triggers the production of Th17-related cytokines. Moreover, we have found that prostaglandin E2 (PGE2) is also produced during efferocytosis containing microbial products, but the effect of this prostanoid on Th17-differentiation is still unknown. The purpose of this study was to evaluate the role of PGE2, produced during recognition of IAC by DC, on Th17 differentiation. As source of IAC, Raw 264-7 cells were cultured with Escherichia coli during 2h for phagocytosis and apoptosis was induced by UV irradiation. BMDC were co-culture with IAC during 18h, and the supernatant from this co-culture was used to differentiate naïve CD4+ T cells for 4d, plus anti-CD3/anti-CD28. Phagocytosis of IAC by DC promotes the production of high levels of PGE2 (15±0.7 ng/ml), IL-6 (26±0.4 ng/ml), TGF-β (3.5±0.09 ng/ml) and IL-1β (5.4±0.1 ng/ml), when compared to phagocytosis of uninfected-AC. The treatment with COX inhibitor significantly decreased PGE2 level and promotes Th17 cell differentiation at least 4 times higher (16%) than untreated co-cultures (4%). Moreover, adding back exogenous PGE2 decreased drastically the percentage of Th17 cells (2%). Moreover, the treatment of naive T cells with antagonists of PGE2 receptor (EP1/EP2/EP4) suggests that the suppressor effect of this prostanoid on Th17 cell differentiation is mainly mediated by EP4/cAMP/PKA activation pathway. Taken all together, our results show that PGE2 is able to suppress Th17 differentiation in vitro through EP4 signaling. Further studies are needed to clarify how PGE2 can modulate the Th17 differentiation using in vivo model. Financial Support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7.

PGE2 imbalance in diabetic mice impairs dendritic cell maturation and favors methicillin-resistant S. aureus skin infection

Abstract Diabetics are more prone to Staphylococcus aureus skin infection than healthy individuals. Neutrophil recruitment and apoptosis are hallmarks of S. aureus infections. Ingestion of infected apoptotic cells (IAC) by dendritic cells (DC) lead to secretion of Th17-related cytokines and prostaglandin E2 (PGE2). We speculate that hyperglycemia inhibits DC migration to the lymph nodes, in a manner dependent on PGE2 levels and impairs Th17 differentiation during MRSA skin infection in type 1 diabetic mice (T1D). Control and T1D mice were subcutaneously (s.c.) infected with 5×106 MRSA CFU. T1D mice showed increased lesion and bacterial load and decreased Th17 population in the skin than nondiabetic mice. We next determined if decreased Th17 correlates with deficient DC migration to draining LN. To track DC migration, mice received s.c. FarRed to stain the skin cells. Our results show that T1D had lower percentage and number of FarRed+DCs+ in the LN than controls.. Impaired DC migration and Th17 differentiation in T1D correlated with decreased skin PGE2 levels. When BMDCs were cultured in high glucose, we observed decreased PGE2 production, migration and maturation after recognition of MRSA-IAC when compared to BMDC kept in normal glucose. Topical treatment with PGE analog (Misoprostol), restored skin DC migration, Th17 differentiation, increased anti-microbial peptides, decreased CFU loads and lesion size in T1D. These data suggest that PGE2 imbalance in hyperglycemia impairs DC migration to LN and fail to mount Th17 responses leading to increased lesions and delayed healing in MRSA skin infections. Misoprostol may be a promising candidate to improve innate and adaptive immune responses in diabetic skin infections.

ENOS and iNOS trigger apoptosis in the brains of sheep and goats naturally infected with the border disease virus.

In this study, apoptotic and anti-apoptotic mechanisms and if present, which pathway to trigger the apoptosis in the brains of Border Disease Virus (BDV) infected lambs (n=10) and goat kids (n=5) were investigated. Briefly, apoptotic (caspase 3, caspase 9) and anti-apoptotic markers (Bcl-2), cytokine response (TNF-α, INF-γ), reactive gliosis and myelin loss were examined. eNOS, iNOS, caspase 9, caspase 3 and GFAP expressions were higher in BDV infected tissues compared to control animals (6 kids and 6 lambs) (p<0.05). Double immunoperoxidase test revealed that TUNEL positive apoptotic cells showed significant association with increased eNOS-iNOS and iNOS-BDV expressions. However, no significant differences were found for TNFR1, TNF-α and INF-γ expressions in BD (p>0.05). There was a positive correlation between the intensity of myelin loss, GFAP activity and severity of infection. Inconclusion, as a novel finding, it is established that eNOS and iNOS overexpressions are co-associated with apoptosis in BDV infected neurons and neuroglia. The results also strongly suggested that BDV infected apoptotic cells mainly prefer the intrinsic pathway that might be most likely related to increased nitric oxide levels.

Cytosolic caspases mediate mislocalised SOD2 depletion in an in vitro model of chronic prion infection

SUMMARYOxidative stress as a contributor to neuronal death during prion infection is supported by the fact that various oxidative damage markers accumulate in the brain during the course of this disease. The normal cellular substrate of the causative agent, the prion protein, is also linked with protective functions against oxidative stress. Our previous work has found that, in chronic prion infection, an apoptotic subpopulation of cells exhibit oxidative stress and the accumulation of oxidised lipid and protein aggregates with caspase recruitment. Given the likely failure of antioxidant defence mechanisms within apoptotic prion-infected cells, we aimed to investigate the role of the crucial antioxidant pathway components, superoxide dismutases (SOD) 1 and 2, in an in vitro model of chronic prion infection. Increased total SOD activity, attributable to SOD1, was found in the overall population coincident with a decrease in SOD2 protein levels. When apoptotic cells were separated from the total population, the induction of SOD activity in the infected apoptotic cells was lost, with activity reduced back to levels seen in mock-infected control cells. In addition, mitochondrial superoxide production was increased and mitochondrial numbers decreased in the infected apoptotic subpopulation. Furthermore, a pan-caspase probe colocalised with SOD2 outside of mitochondria within cytosolic aggregates in infected cells and inhibition of caspase activity was able to restore cellular levels of SOD2 in the whole unseparated infected population to those of mock-infected control cells. Our results suggest that prion propagation exacerbates an apoptotic pathway whereby mitochondrial dysfunction follows mislocalisation of SOD2 to cytosolic caspases, permitting its degradation. Eventually, cellular capacity to maintain oxidative homeostasis is overwhelmed, thus resulting in cell death.

Role for Rab11a in mediating TLR dependent cross-presentation responses (106.17)

Abstract Toll-like receptor (TLR) signaling is critical for accelerated phagosome maturation and enhanced major histocompatibility complex class II (MHC II) presentation of phagocytosed antigens. Since cross-presentation is another consequence of phagosome maturation, we hypothesized that like MHC II presentation, the presentation of exogenous antigens on MHC class I (MHC I) by dendritic cells (DCs) is positively regulated by TLRs. Using antigen derived from bacteria, infected apoptotic cells or antigen-coated beads conjugated to a TLR ligand, our results show that cross-presentation is severely impaired when TLR signaling is abrogated in the cross-presenting DC or when TLR ligands are absent from phagocytic cargo. To delineate the mechanisms behind TLR enhancement of cross-presentation, we studied trafficking of MHC I in the presence or absence of TLR signaling. We observed recruitment of MHC I selectively to phagosomes containing TLR ligands along with the concomitant enrichment of the small guanosine tri-phosphatase (GTPase) Rab11a. In fact, Rab11a was crucial for TLR mediated enhancement of cross-presentation as selective knock-down of Rab11a abrogated the ability of DCs to cross-present antigen. Our results demonstrate a role for Rab11a in trafficking MHC I to phagosomes that engage TLR signaling, thus mediating enhanced cross-presentation of microbial antigens. Results from our studies have implications on anti-viral and anti-bacterial immunity as well as tumor immunotherapy.

ORF3 of porcine circovirus 2 enhances the in vitro and in vivo spread of the of the virus

The ORF3 protein of the pathogenic porcine circovirus 2 (PCV2) causes apoptosis of the virus-infected cells. In PCV2-infected piglets, ORF3 induces B and CD4 T lymphocyte depletion and lymphoid organ destruction and the ORF3-deficient PCV2 is attenuated in its pathogenicity ( Virology, 383 (2009), 338). In addition to its role in causing the apoptosis of the immune cells, characteristic of the PCV2 infection associated disease conditions, the ORF3 also plays a role in the systemic dissemination of the PCV2 infection. Our experiments here show that ORF3 expedites the spread of the virus by inducing the early release of the virus from the infected cells. Further, in PCV2-infected mice, the ORF3-induced apoptosis also aids in recruiting macrophages to phagocytize the infected apoptotic cells leading to the systemic dissemination of the infection. The apoptotic activity of the ORF3 of PCV2 hence lends advantage to the spread of the virus.

Innate immune recognition of infected apoptotic cells directs TH17 cell differentiation

Adaptive immune responses rely on differentiation of CD4 T helper cells into subsets with distinct effector functions best suited for host defence against the invading pathogen. Interleukin (IL)-17-producing T helper cells (T(H)17) are a recently identified subset, separate from the T helper type 1 (T(H)1) and T helper type 2 (T(H)2) subsets. Synergy between the cytokines transforming growth factor-beta and IL-6 in vitro induces development of T(H)17 cells in mouse and human systems, whereas IL-23 supports expansion of these cells. However, it is not known which conditions in vivo would induce this combination of cytokines. Furthermore, it is enigmatic that a combination of pro-inflammatory and anti-inflammatory cytokines would be required to generate an effector T(H)17 response. Here we show that the relevant physiological stimulus triggering this combination of cytokines is the recognition and phagocytosis of infected apoptotic cells by dendritic cells. Phagocytosis of infected apoptotic cells uniquely triggers the combination of IL-6 and transforming growth factor-beta through recognition of pathogen-associated molecular patterns and phosphatidylserine exposed on apoptotic cells, respectively. Conversely, phagocytosis of apoptotic cells in the absence of microbial signals induces differentiation of the closely related regulatory T cells, which are important for controlling autoimmunity. Blocking apoptosis during infection of the mouse intestinal epithelium with the rodent pathogen Citrobacter rodentium, which models human infections with the attaching and effacing enteropathogenic and enterohaemorrhagic Escherichia coli, impairs the characteristic T(H)17 response in the lamina propria. Our results demonstrate that infected apoptotic cells are a critical component of the innate immune signals instructing T(H)17 differentiation, and point to pathogens particularly adept at triggering apoptosis that might preferentially induce T(H)17-mediated immunity. Because T(H)17 cells have been correlated with autoimmune diseases, investigation of the pathways of innate recognition of infected apoptotic cells might lead to improved understanding of the causative defects in autoimmunity.

Hepatitis C virus infected apoptotic cells (bodies) differentially activate hepatic Stellate cells as compared to non-infected apoptotic bodies

Hepatitis C virus infected apoptotic cells (bodies) differentially activate hepatic Stellate cells as compared to non-infected apoptotic bodies

Synthetic Vpr Protein Activates Activator Protein-1, c-Jun N-terminal Kinase, and NF-κB and Stimulates HIV-1 Transcription in Promonocytic Cells and Primary Macrophages

The human immunodeficiency virus (HIV) Vpr protein plays a critical role in AIDS pathogenesis, especially by allowing viral replication within nondividing cells such as mononuclear phagocytes. Most of the data obtained so far have been in experiments with endogenous Vpr protein; therefore the effects of extracellular Vpr protein remain largely unknown. We used synthetic Vpr protein to activate nuclear transcription factors activator protein-1 (AP-1) and NF-kappaB in the promonocytic cell line U937 and in primary macrophages. Synthetic HIV-1 Vpr protein activated AP-1, c-Jun N-terminal kinase, and MKK7 in both U937 cells and primary macrophages. Synthetic Vpr activated NF-kappaB in primary macrophages and to a lesser extent in U937 cells. Because synthetic Vpr activated AP-1 and NF-kappaB, which bind to the HIV-1 long terminal repeat, we investigated the effect of synthetic Vpr on HIV-1 replication. We observed that synthetic Vpr stimulated HIV-1 long terminal repeat in U937 cells and enhanced viral replication in chronically infected U1 promonocytic cells. Similarly, synthetic Vpr stimulated HIV-1 replication in acutely infected primary macrophages. Activation of transcription factors and enhancement of viral replication in U937 cells and primary macrophages were mediated by both the N-terminal and the C-terminal moieties of synthetic Vpr. Therefore, our results suggest that extracellular Vpr could fuel the progression of AIDS via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.

INCREASED APOPTOSIS AND EXPRESSION OF TUMOR NECROSIS FACTOR-α CAUSED BY INFECTION OF CULTURED HUMAN MONOCYTES WITH DENGUE VIRUS

Dengue (DEN) virus is responsible for one of the most significant viral diseases in tropical countries. Monocytes/macrophages (Mo/Mphi) are the major target cells for DEN virus. To determine the effects of the interaction between DEN virus and Mo/Mphi, human monocyte cultures were infected with DEN virus type 2. Apoptosis and production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured in control and infected cultures. Virus was taken up by phagocytosis, but no membrane-coated pits at the virus attachment sites were observed. Increased number of apoptotic cells and increased production of TNF-a were observed in infected monocyte cultures. No increase in production of nitric oxide was observed. These results may be related to early primary viral infection, in which virus could induce apoptosis in monocytes, but monocytes may contribute to host defense mechanisms against virus by viral phagocytosis, phagocytosis of infected apoptotic cells, and the release of proinflammatory cytokines.

Efficiency of cross presentation of vaccinia virus-derived antigens by human dendritic cells.

Dendritic cells (DC) utilize at least two pathways to process viral antigens onto MHC class I molecules. The conventional endogenous route is used to acquire antigens from both infectious and non-replicating virions. Exogenous pathways are used by DC to acquire and "cross-present" antigens derived from virus-infected donor cells that by themselves lack the ability to activate T cells directly. We analyzed the role of this pathway for antigens derived from vaccinia, a virus which inhibits DC maturation and causes extensive apoptosis of infected cells, yet is highly immunogenic. Using recombinant vaccinia virus encoding the influenza matrix protein as model vector, DC were shown to cross-present vaccinia-derived antigens from both apoptotic and necrotic infected cells to antigen-specific CD8(+) T cells. Efficient cross presentation required uptake of dead cells by immature DC and exposure to maturation stimuli, especially CD40 ligand. The responding CD8(+) T cells secreted IL-2 and IFN-gamma, proliferated and developed into cytotoxic effectors. Quantification of the cross presentation of vaccinia-derived antigens showed this pathway to be highly efficient, corresponding to a peptide pulse of 10-100 nM. While monocytes also phagocytosed apoptotic and necrotic cells, they were far less efficient at cross-presenting vaccinia-derived antigens to CD8(+) T cells. The ability of DC to cross-present vaccinia-derived antigens from infected apoptotic cells or necrotic cell lysates, bypasses the deleterious effects of direct infection of DC and provides one explanation for this pathogen's immunogenicity.