GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I

Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19(-/-) blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development.     

The uncoupling protein 2 modulates the cytokine balance in innate immunity

The uncoupling protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.     

Innate immune responses to Rhodococcus equi

We examined innate immune responses to the intracellular bacterium Rhodococcus equi and show that infection of macrophages with intact bacteria induced the rapid translocation of NF-kappa B and the production of a variety of proinflammatory mediators, including TNF, IL-12, and NO. Macrophages from mice deficient in MyD88 failed to translocate NF-kappa B and produced virtually no cytokines in response to R. equi infection, implicating a TLR pathway. TLR4 was not involved in this response, because C3H/HeJ macrophages were fully capable of responding to R. equi infection, and because RAW-264 cells transfected with a dominant negative form of TLR4 responded normally to infection by R. equi. A central role for TLR2 was identified. A TLR2 reporter cell was activated by R. equi, and RAW-264 cells transfected with a dominant negative TLR2 exhibited markedly reduced cytokine responses to R. equi. Moreover, macrophages from TLR2(-/-) mice exhibited diminished cytokine responses to R. equi. The role of the surface-localized R. equi lipoprotein VapA (virulence-associated protein A), in TLR2 activation was examined. Purified rVapA activated a TLR2-specific reporter cell, and it induced the maturation of dendritic cells and the production of cytokines from macrophages. Importantly, TLR2(-/-)-deficient but not TLR4(-/-)-deficient mice were found to be compromised in their ability to clear a challenge with virulent R. equi. We conclude that the efficient activation of innate immunity by R. equi may account for the relative lack of virulence of this organism in immunocompetent adults.     

STAT4 is a critical mediator of early innate immune responses against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of morbidity and mortality in the U.S. An effective innate immune response is critical for the clearance of bacteria from the lungs. IL-12, a key T1 cytokine in innate immunity, signals through STAT4. Thus, understanding how STAT4 mediates pulmonary immune responses against bacterial pathogens will have important implications for the development of rational immunotherapy targeted at augmenting innate immunity. We intratracheally administered Klebsiella pneumoniae to wild-type BALB/c and STAT4 knockout (STAT4-/-) mice. Compared with wild-type controls, STAT4-/- mice had decreased survival following intratracheal Klebsiella administration, which was associated with a higher lung and blood bacterial burden. STAT4-/- animals also displayed impaired pulmonary IFN-gamma production and decreased levels of proinflammatory cytokines, including the ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma. Although total lung leukocyte populations were similar between STAT4-/- and wild-type animals following infection, alveolar macrophages isolated from infected STAT4-/- mice had decreased production of proinflammatory cytokines, including IFN-gamma, compared with infected wild-type mice. The intrapulmonary overexpression of IFN-gamma concomitant with the systemic administration of IFN-gamma partially reversed the immune deficits observed in STAT4-/- mice, resulting in improved bacterial clearance from the blood. Collectively, these studies demonstrate that STAT4 is required for the generation of an effective innate host defense against bacterial pathogens of the lung.     

GRIM-19 is essential for maintenance of mitochondrial membrane potential

GRIM-19 was found to copurify with complex I of mitochondrial respiratory chain and subsequently was demonstrated to be involved in complex I assembly and activity. To further understand its function in complex I, we dissected its functional domains by generating a number of deletion, truncation, and point mutants. The mitochondrial localization sequences were located at the N-terminus. Strikingly, deletion of residues 70-80, 90-100, or the whole C-terminal region (70-144) led to a loss of mitochondrial transmembrane potential (DeltaPsim). However, similar deletions of another two complex I subunits, NDUFA9 and NDUFS3, did not show such effect. We also found that deletion of the last 10 residues affected GRIM-19's ability to be assembled to complex I. We constructed a dominant-negative mutant containing the N-terminal 60 and the last C-terminal 10 residues, which could be assembled into complex I, but failed to maintain normal DeltaPsim. Cells overexpressing this mutant did not spontaneously undergo cell death, but were sensitized to apoptosis induced by cell death agents. Our results demonstrate that GRIM-19 is required for electron transfer activity of complex I, and disruption of DeltaPsim by GRIM-19 mutants enhances the cells' sensitivity to apoptotic stimuli.     

Vdelta1+ gammadelta T cells producing CC chemokines may bridge a gap between neutrophils and macrophages in innate immunity during Escherichia coli infection in mice

An influx of neutrophils followed a short time later by an influx of macrophages to the infected site plays a key role in innate immunity against Escherichia coli infection. We found in this study that Vdelta1-/- mice exhibited impaired accumulation of peritoneal macrophages but not neutrophils and delayed bacterial clearance after i.p. inoculation with E. coli. Peritoneal gammadelta T cells from E. coli-infected wild-type mice produced CCL3/MIP-1alpha and CCL5/RANTES in response to gammadelta TCR triggering in vitro, whereas such production was not evident in gammadelta T cells from E. coli-infected Vdelta1-/- mice. Neutralization of CCL3/MIP-1alpha by a specific mAb in vivo significantly inhibited the accumulation of macrophages in the peritoneal cavity after E. coli infection, resulting in exacerbated bacterial growth in the peritoneal cavity. These results suggest that Vdelta1+ gammadelta T cells bridge a gap between neutrophils and macrophages in innate immunity during E. coli infection mediated by production of CC chemokines, enhancing macrophage trafficking to the site of infection.     

Th1 and Th17 cells regulate innate immune responses and bacterial clearance during central nervous system infection

Brain abscesses arise following parenchymal infection with pyogenic bacteria and are typified by inflammation and edema, which frequently results in a multitude of long-term health problems. The impact of adaptive immunity in shaping continued innate responses during late-stage brain abscess formation is not known but is important, because robust innate immunity is required for effective bacterial clearance. To address this issue, brain abscesses were induced in TCR αβ knockout (KO) mice, because CD4(+) and NKT cells represented the most numerous T cell infiltrates. TCR αβ KO mice exhibited impaired bacterial clearance during later stages of infection, which was associated with alterations in neutrophil and macrophage recruitment, as well as perturbations in cytokine/chemokine expression. Adoptive transfer of either Th1 or Th17 cells into TCR αβ KO mice restored bacterial burdens and innate immune cell infiltrates to levels detected in wild-type animals. Interestingly, adoptively transferred Th17 cells demonstrated plasticity within the CNS compartment and induced distinct cytokine secretion profiles in abscess-associated microglia and macrophages compared with Th1 transfer. Collectively, these studies identified an amplification loop for Th1 and Th17 cells in shaping established innate responses during CNS infection to maximize bacterial clearance and differentially regulate microglial and macrophage secretory profiles.     

Suppression of Mitochondrial Complex I Influences Cell Metastatic Properties

Despite the fact that mitochondrial dysfunction has an important role in tumorigenesis and metastasis, the underlying mechanism remains to be elucidated. Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) is the first and the largest protein complex of the mitochondrial electron-transport chain (ETC),which has an essential role in maintaining mitochondrial function and integrity. In this study, we separately knocked down two subunits of mitochondrial complex I, GRIM-19 or NDUFS3, and investigated their effects on metastatic behaviors and explored the possible mechanisms. Our data showed that stable down-modulation of GRIM-19 or NDUFS3 decreased complex I activity and reactive oxygen species (ROS) production; led to enhanced cell adhesion, migration, invasion, and spheroid formation; and influenced the expressions of extracellular matrix (ECM) molecules and its related proteins. We also observed that the expressions of GRIM-19, NDUFS3, and ECM elements were correlated with invasive capabilities of breast cancer cell lines. These results suggest that inhibition of complex I affects metastatic properties of cancer cells, and mitochondrial ROS might play a crucial role in these processes by regulating ECM.     

Overexpression of suppressor of cytokine signaling-5 in T cells augments innate immunity during septic peritonitis

Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT signal transduction pathway, but their role in innate immunity remains to be investigated. In the present study, we demonstrate that overexpression of SOCS5 in T cells augments innate immunity during septic peritonitis induced by cecal ligation and puncture (CLP). Mice with a cell-specific overexpression of SOCS5 in T cells (SOCS5 transgenic (Tg)) were resistant to the lethality relative to the wild-type (WT) mice. This was most likely due to the enhanced innate immunity in SOCS5Tg mice, as bacterial burden in SOCS5Tg mice was significantly lower than WT mice. Accumulation of neutrophils and macrophages was augmented in SOCS5Tg mice, an event that was accompanied by increased peritoneal levels of IL-12, IFN-gamma, and TNF-alpha. In vitro bactericidal activities of macrophages and neutrophils were enhanced in SOCS5Tg mice. Both neutrophils and macrophages from WT mice adopted enhanced bacterial killing activity when cocultured with CD4+ T cells from SOCS5Tg mice, relative to CD4+ T cells from WT mice. Adoptive transfer of SOCS5Tg-CD4+ T cells into T- and B cell-deficient RAG-2(-/-) mice resulted in augmented leukocyte infiltration and increased peritoneal levels of IL-12, IFN-gamma, and TNF-alpha after CLP, as compared with the controls. Furthermore, CLP-induced bacterial burden in RAG-2(-/-) mice harboring SOCS5Tg-CD4+ T cells was significantly reduced relative to the controls. These findings provide evidence that intervention of SOCS5 expression in T cells affects innate immunity, which highlight a novel role of T cells during sepsis.     

Expression and functional characterization of a gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) from orange-spotted grouper (Epinephelus coioides)

GRIM-19 is a nuclear encoded subunit of complex I that has been implicated in apoptosis. The protein participates in multiple functions including the innate immune response. GRIM-19 has been studied in humans and other mammals; however, fish GRIM-19 has not been well characterized. In this study, a new GRIM-19 gene, EcGRIM-19, was isolated from the orange-spotted grouper (Epinephelus coioides) cDNA library, which was constructed following LPS treatment. EcGRIM-19 is a 582-bp gene that encodes a 144-amino acid protein. The gene is a true ortholog of mammalian GRIM-19. EcGRIM-19 exhibits ubiquitous and constitutive expression in the different tissues of the orange-spotted grouper. The expression levels of EcGRIM-19 are altered in the gill, spleen, kidney and liver after induction with LPS. The subcellular localization analysis demonstrated that the EcGRIM-19 protein is localized predominantly in the mitochondria. In addition, amino acids 30–50 of the protein are responsible for the mitochondrial localization of EcGRIM-19. The caspase assay demonstrated that the overexpression of GRIM-19 enhanced the cellular sensitivity to interferon(IFN)-β- and retinoic acid (RA)-induced death in HeLa cells. The data presented in this study are important for further understanding the EcGRIM-19 gene function in fish.     

Infection with the Lyme disease pathogen suppresses innate immunity in mice with diet‐induced obesity

Obesity is a major global public health concern. Immune responses implicated in obesity also control certain infections. We investigated the effects of high‐fat diet‐induced obesity (DIO) on infection with the Lyme disease bacterium Borrelia burgdorferi in mice. DIO was associated with systemic suppression of neutrophil‐ and macrophage‐based innate immune responses. These included bacterial uptake and cytokine production, and systemic, progressive impairment of bacterial clearance, and increased carditis severity. B. burgdorferi‐infected mice fed normal diet also gained weight at the same rate as uninfected mice fed high‐fat diet, toll‐like receptor 4 deficiency rescued bacterial clearance defects, which greater in female than male mice, and killing of an unrelated bacterium (Escherichia coli) by bone marrow‐derived macrophages from obese, B. burgdorferi‐infected mice was also affected. Importantly, innate immune suppression increased with infection duration and depended on cooperative and synergistic interactions between DIO and B. burgdorferi infection. Thus, obesity and B. burgdorferi infection cooperatively and progressively suppressed innate immunity in mice.     

Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus

Myeloid differentiation factor 88 (MyD88) is an adapter molecule required for signal transduction via Toll-like receptors (TLRs) and receptors of the IL-1 family. Consequently, MyD88-deficient mice are highly susceptible to bacterial infections, including systemic infection with Staphylococcus aureus. To determine the role of MyD88 in innate immunity to bacterial pneumonia, we exposed MyD88-deficient and wild-type mice to aerosolized Pseudomonas aeruginosa or S. aureus. As predicted, MyD88-deficient mice failed to mount an early cytokine or inflammatory response or to control bacterial replication after infection with P. aeruginosa, which resulted in necrotizing pneumonia and death. By contrast, MyD88-deficient mice controlled S. aureus infection despite blunted local cytokine and inflammatory responses. Thus, whereas MyD88-dependent signaling is integral to the initiation of cytokine and inflammatory responses to both pathogens following infection of the lower respiratory tract, MyD88 is essential for innate immunity to P. aeruginosa but not S. aureus.     

Innate Immune Dysfunctions in Aged Mice Facilitate the Systemic Dissemination of Methicillin-Resistant S. aureus

Elderly humans show increased susceptibility to invasive staphylococcal disease after skin and soft tissue infection. However, it is not understood how host immunity changes with aging, and how that predisposes to invasive disease. In a model of severe skin infection, we showed that aged mice (16- to 20-month-old) exhibit dramatic bacterial dissemination compared with young adult mice (2-month-old). Bacterial dissemination was associated with significant reductions of CXCL1 (KC), polymorphonuclear cells (PMNs), and extracellular DNA traps (NETs) at the infection site. PMNs and primary skin fibroblasts isolated from aged mice showed decreased secretion of CXCL2 (MIP-2) and KC in response to MRSA, and in vitro analyses of mitochondrial functions revealed that the mitochondrial electron transport chain complex I plays a significant role in induction of chemokines in the cells isolated from young but not old mice. Additionally, PMNs isolated from aged mice have reduced ability to form NETs and to kill MRSA. Expression of nuclease by S. aureus led to increased bacterial systemic dissemination in young but not old mice, suggesting that defective NETs formation in elderly mice permitted nuclease and non-nuclease expressing S. aureus to disseminate equally well. Overall, these findings suggest that gross impairment of both skin barrier function and innate immunity contributes to the propensity for MRSA to disseminate in aged mice. Furthermore, the study indicates that contribution of bacterial factors to pathogenicity may vary with host age.     

Expression of the p60 autolysin enhances NK cell activation and is required for listeria monocytogenes expansion in IFN-gamma-responsive mice

Both peptidoglycan and muropeptides potently modulate inflammatory and innate immune responses. The secreted Listeria monocytogenes p60 autolysin digests peptidoglycan and promotes bacterial infection in vivo. Here, we report that p60 contributes to bacterial subversion of NK cell activation and innate IFN-gamma production. L. monocytogenes deficient for p60 (Deltap60) competed well for expansion in mice doubly deficient for IFNAR1 and IFN-gammaR1 or singly deficient for IFN-gammaR1, but not in wild-type, IFNAR1(-/-), or TLR2(-/-) mice. The restored competitiveness of p60-deficient bacteria suggested a specific role for p60 in bacterial subversion of IFN-gamma-mediated immune responses, since in vivo expansion of three other mutant L. monocytogenes strains (DeltaActA, DeltaNamA, and DeltaPlcB) was not complemented in IFN-gammaR1(-/-) mice. Bacterial expression of p60 was not required to induce socs1, socs3, and il10 expression in infected mouse bone marrow macrophages but did correlate with enhanced production of IL-6, IL-12p70, and most strikingly IFN-gamma. The primary source of p60-dependent innate IFN-gamma was NK cells, whereas bacterial p60 expression did not significantly alter innate IFN-gamma production by T cells. The mechanism for p60-dependent NK cell stimulation was also indirect, given that treatment with purified p60 protein failed to directly activate NK cells for IFN-gamma production. These data suggest that p60 may act on infected cells to indirectly enhance NK cell activation and increase innate IFN-gamma production, which presumably promotes early bacterial expansion through its immunoregulatory effects on bystander cells. Thus, the simultaneous induction of IFN-gamma production and factors that inhibit IFN-gamma signaling may be a common strategy for misdirection of early antibacterial immunity.     

Role of specific innate immune responses in herpes simplex virus infection of the central nervous system

Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.     

DNA-PKcs is required for activation of innate immunity by immunostimulatory DNA

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribonucleotides (ISS-ODN) stimulate innate immunity. However, the molecular recognition mechanism that initiates signaling in response to bacterial DNA and ISS-ODN has not been identified. Herein, we demonstrate that administration of bacterial DNA and ISS-ODN to mice lacking the catalytic subunit of DNA-PK (DNA-PKcs) and in vitro stimulation of BMDM from these mice result in defective induction of IL-6 and IL-12. Further analysis using BMDM of IKKbeta(-/-) revealed that both DNA-PKcs and IKKbeta are essential for normal cytokine production in response to ISS-ODN or bacterial DNA. ISS-ODN and bacterial DNA activate DNA-PK, which in turn contributes to activation of IKK and NF-kappaB. These results reveal a novel role of DNA-PKcs in innate immune responses and a link between DNA repair and innate immunity.