Herpes simplex virus 1 (HSV-1) can undergo a productive infection in nonneuronal and neuronal cells such that the genes of the virus are transcribed in an ordered cascade. HSV-1 can also establish a ...more quiescent or latent infection in peripheral neurons, where gene expression is substantially reduced relative to that in productive infection. HSV mutants defective in multiple immediate early (IE) gene functions are highly defective for later gene expression and model some aspects of latency in vivo. We compared the expression of wild-type (wt) virus and IE gene mutants in nonneuronal cells (MRC5) and adult murine trigeminal ganglion (TG) neurons using the Illumina platform for cDNA sequencing (RNA-seq). RNA-seq analysis of wild-type virus revealed that expression of the genome mostly followed the previously established kinetics, validating the method, while highlighting variations in gene expression within individual kinetic classes. The accumulation of immediate early transcripts differed between MRC5 cells and neurons, with a greater abundance in neurons. Analysis of a mutant defective in all five IE genes (d109) showed dysregulated genome-wide low-level transcription that was more highly attenuated in MRC5 cells than in TG neurons. Furthermore, a subset of genes in d109 was more abundantly expressed over time in neurons. While the majority of the viral genome became relatively quiescent, the latency-associated transcript was specifically upregulated. Unexpectedly, other genes within repeat regions of the genome, as well as the unique genes just adjacent the repeat regions, also remained relatively active in neurons. The relative permissiveness of TG neurons to viral gene expression near the joint region is likely significant during the establishment and reactivation of latency.
During productive infection, the genes of HSV-1 are transcribed in an ordered cascade. HSV can also establish a more quiescent or latent infection in peripheral neurons. HSV mutants defective in multiple immediate early (IE) genes establish a quiescent infection that models aspects of latency in vivo. We simultaneously quantified the expression of all the HSV genes in nonneuronal and neuronal cells by RNA-seq analysis. The results for productive infection shed further light on the nature of genes and promoters of different kinetic classes. In quiescent infection, there was greater transcription across the genome in neurons than in nonneuronal cells. In particular, the transcription of the latency-associated transcript (LAT), IE genes, and genes in the unique regions adjacent to the repeats persisted in neurons. The relative activity of this region of the genome in the absence of viral activators suggests a more dynamic state for quiescent genomes persisting in neurons.
Hepatocytes play a crucial role in host response to infection.
Ehrlichia
is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) ...characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent
Ehrlichia japonica
(
E. japonica
) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for
Ehrlichia
, the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with
E. japonica
using primary cells from wild-type (WT) and MyD88
-/-
mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in
Ehrlichia
-infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88
-/-
HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in
E. japonica
-infected WT, but not infected MyD88
-/-
HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during
Ehrlichia
infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as
Ehrlichia
.
Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We ...have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) ...infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background: HCC is one of the most lethal cancers for humans. Mannosidase alpha class 2A member 1 (MAN2A1)-FER is one of the most frequent oncogenic fusion genes in HCC. In this report, we showed ...that MAN2A1-FER ectopically phosphorylated the extracellular domains of PDGFRA, MET, AXL, and N-cadherin. The ectopic phosphorylation of these transmembrane proteins led to the activation of their kinase activities and initiated the activation cascades of their downstream signaling molecules. Methods: A panel of mouse monoclonal antibodies was developed to recognize the ectopic phosphorylation sites of PDGFRA. Results and conclusions: The analyses showed that these antibodies bound to the specific phosphotyrosine epitopes in the extracellular domain of PDGFRA with high affinity and specificity. The treatment of MAN2A1-FER–positive cancer HUH7 with one of the antibodies called 2-3B-G8 led to the deactivation of cell growth signaling pathways and cell growth arrest while having minimal impact on HUH7ko cells where MAN2A1-FER expression was disrupted. The treatment of 2-3B-G8 antibody also led to a large number of cell deaths of MAN2A1-FER–positive cancer cells such as HUH7, HEPG2, SNU449, etc., while the same treatment had no impact on HUH7ko cells. When severe combined immunodeficiency mice xenografted with HEPG2 or HUH7 were treated with monomethyl auristatin E-conjugated 2-3B-G8 antibody, it slowed the progression of tumor growth, eliminated the metastasis, and reduced the mortality, in comparison with the controls. Targeting the cancer-specific ectopic phosphorylation sites of PDGFRA induced by MAN2A1-FER may hold promise as an effective treatment for liver cancer.
Background
Liver is the major target organ in Human Monocytic ehrlichiosis (HME), an undifferentiated febrile illness that is life‐threatening and caused by intracellular bacterial pathogen, ...Ehrlichia, that target liver and infect macrophages, hepatocytes and endothelial cells. Severe Hepatic inflammation is the common cause of acute liver injury following systemic infection with Ehrlichia. Our previous studies have shown that Myeloid differentiation primary response gene 88 (MYD88) and type I IFN (IFN‐I) promotes excessive inflammation and liver damage following fatal Ehrlichia infection. In this study, we explored the role of the MyD88 and IFN‐I in liver inflammation.
Results
In this study, we provide evidence to support that high mobility group box 1 (HMGB1), a chromosomal protein and damage‐associated molecular patterns (DAMPs), plays a role in the regulation of innate responses and autophagy during Ehrlichia infection. Wild‐type (WT) mice infected with virulent Ehrlichia develop sepsis and liver injury marked by excessive systemic production of pro‐inflammatory cytokines and chemokines as well as HMGB1. To determine the cellular source of HMGB1 and regulatory mechanisms, we infected hepatocytes (HC) from WT and MYD88−/− knockout mice with virulent Ehrlichia. To mimic paracrine effect of IFN‐I cytokines produced by immune cells on HC response to Ehrlichia infection, we cultured infected HC in the presence or absence of IFN‐β (100 IU/mL). Our data indicate that in‐vitro infected HC produces low levels of IFN‐beta compared to uninfected cells. Exogenous stimulation of infected HC with IFN‐β induced elevated expression and cytoplasmic translocation of HMGB1 in WT‐HC. This was associated with increased autophagy and higher expression of IL‐23R, IFNAR, and heightened JAK/STAT signaling. Importantly, the effect of high dose IFN‐I on autophagy and HMGB1 was MYD88‐dependent. Interestingly, infection of MYD88−/− HC with Ehrlichia, in the absence of exogenous IFN‐β, enhanced autophagy induction and cytosolic translocation of HMGB1, suggesting that MYD88 negatively regulate HMGB1 and autophagy in HC in the absence of the paracrine effect of IFN‐β.
Conclusion
Together, our data suggest that autophagy regulation and HMGB1 response in hepatocytes during Ehrlichia‐induced sepsis is controlled by the strength of IFN‐I signaling and dependent on interplay between MYD88 and IFN‐I pathways.
Support or Funding Information
This work was supported by grants from NIAMS, NIGMD, and NIAID (AR68317 CCY, RO1‐GM102146 MS, and R56A1097679 NI)
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few—if any—germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that ...express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.
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•E. muris induces localized B cell proliferation, differentiation, and SHM in liver•Primary response and MBC clones interchange between liver and spleen•E. muris induces T-bet+ MBCs that adopt follicular, ABC, and MZ phenotypes•E. muris dissolves the MZ, which is subsequently reconstituted by T-bet+ MBCs
Infection by the intracellular bacterium Ehrlichia induces few—if any—germinal centers, yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs). Trivedi et al. show that the liver and spleen are generative sites of B cell responses, including V-region mutation and long-term MBC localization, to E. muris.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Macrophages heterogeneity that undergo dynamic changes in phenotype and function as well as reprograming of immune response to multiple intracellular bacterial infections is an important key ...to understand the host responses to infection. Ehrlichia is an obligate Gram-negative intracellular bacterium that cause potentially fatal human monocytotropic ehrlichiosis (HME). We utilized murine models of mild and fatal ehrlichiosis caused by Ehrlichia muris and Ixodes Ovatus Ehrlichia (IOE), respectively, to evaluate the macrophages polarization to inflammatory (M1) and regenerative (M2). Our data show that in vivo and in vitro infections of macrophages with IOE significantly polarize macrophages towards M1, while EM infection enhances M2 polarization. M1 polarization of macrophages in IOE-infected mice was associated with recruitment and/or expansion of granzyme B+ neutrophils, NK cells, IL-17+ NKT cells, and IL-17+ CD4 Th17 cells compared to EM infection. We previously identified mTORC1 (mammalian target of rapamycin complex-1), a negative regulator of autophagy and the key mediator for immunopathology during IOE infection. We found that M1 polarization in the livers of IOE-infected mice is dependent on mTORC1 activation. Blockade of mTORC1 activation with rapamycin decreased frequency of Th17, and enhanced autophagy, which in turn decreased inflammation as well as the pathogenic immune response during lethal IOE infection. Together, these data reveal a key role of mTORC1 in M1 polarization of macrophages, which likely promotes Ehrlichia-induced sepsis. Understanding the dynamic changes of macrophage polarization and how it affects immune responses to infection is crucial for development novel therapeutic strategies.
Passive immunization is an important parameter of post exposure rabies prophylaxis. Two types of rabies immunoglobulin (RIG) are currently available for Passive immunization against rabies i.e human ...rabies immunoglobulin (HRIG) and equine rabies immunoglobulin (ERIG). The former is very expensive and not easily available and the latter causes side effects because of which its utility is limited. In the present study we have produced murine monoclonal antibodies (Mabs) to rabies glycoprotein (G) and studied their utility in passive immunization against rabies using animal models. Their efficacy was compared to commercially available ERIG both in terms of neutralizing titer and effective protein concentration. The neutralizing titers of these Mabs ranged from 1650 IU/mL to 75,000 IU/mL by RFFIT. They belonged to the IgG 2 a subclass. The Mabs were able to protect 70 to 100% of mice and guinea pigs inoculated with rabies viruses, depending on the strain of the virus. These Mabs were found to be 2000 time more potent than commercial ERIG in terms of effective protein concentration and neutralizing titer. Further studies are required to study their utility in humans exposed to rabies.
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