Role of human metapneumovirus glycoprotein G in modulation of immune responses.

Human metapneumovirus (hMPV) is an important pathogen responsible for acute respiratory tract infections in children, the elderly, and immunocompromised patients, with no effective treatment or vaccine currently available. Knowledge of virus- and host-specific mechanisms contributing to the pathogenesis of hMPV infection is still limited. Studies have shown that hMPV surface glycoprotein G is an important virulence factor, by inhibiting innate immune signaling in airway epithelial cells and immune cells. In this study, we investigated the role of G protein in modulating innate and adaptive immune responses in mice infected with a recombinant virus with deletion of G protein (rhMPV-ΔG). Results show that rhMPV-ΔG was strongly attenuated, as it did not induce significant clinical disease, airway obstruction and airway hyperresponsiveness (AHR), compared to infection with a control strain (rhMPV-WT). By analysis of cells in bronchoalveolar fluid and lung tissue, as well as cytokine production, we found that G protein mediates aspects of both innate and adaptive immune responses, including neutrophils, dendritic cells, natural killer cells and B cells. Lung T cells recruited in response to rhMPV-ΔG had a significantly higher activated phenotype compared to those present after rhMPV-WT infection. Despite highly attenuation characterized by low levels of replication in the lung, rhMPV-ΔG was able to induce neutralizing antibodies and to protect mice from a secondary hMPV challenge. However, challenged mice that had received rhMPV-ΔG as primary infection showed some signs of lung disease at the earliest time points, which were less evident in mice that had received the rhMPV-WT strain as primary infection. These results demonstrate some of the mechanisms by which G protein could contribute to airway disease and modulate immune response to hMPV infection.

Ehrlichia SLiM Ligand Mimetic Activates Notch Signaling in Human Monocytes.

Ehrlichia chaffeensis evades innate host defenses by reprogramming the mononuclear phagocyte through mechanisms that involve the exploitation of multiple evolutionarily conserved cellular signaling pathways, including Notch. This immune evasion strategy is directed in part by tandem repeat protein (TRP) effectors. Specifically, the TRP120 effector activates and regulates Notch signaling through interactions with the Notch receptor and the negative regulator, F-Box and WD repeat domain-containing 7 (FBW7). However, the specific molecular interactions and motifs required for E. chaffeensis TRP120-Notch receptor interaction and activation have not been defined. To investigate the molecular basis of TRP120 Notch activation, we compared TRP120 with endogenous canonical/noncanonical Notch ligands and identified a short region of sequence homology within the tandem repeat (TR) domain. TRP120 was predicted to share biological function with Notch ligands, and a function-associated sequence in the TR domain was identified. To investigate TRP120-Notch receptor interactions, colocalization between TRP120 and endogenous Notch-1 was observed. Moreover, direct interactions between full-length TRP120, the TRP120 TR domain containing the putative Notch ligand sequence, and the Notch receptor LBR were demonstrated. To molecularly define the TRP120 Notch activation motif, peptide mapping was used to identify an 11-amino acid short linear motif (SLiM) located within the TRP120 TR that activated Notch signaling and downstream gene expression. Peptide mutants of the Notch SLiM or anti-Notch SLiM antibody reduced or eliminated Notch activation and NICD nuclear translocation. This investigation reveals a novel molecularly defined pathogen encoded Notch SLiM mimetic that activates Notch signaling consistent with endogenous ligands. IMPORTANCE E. chaffeensis infects and replicates in mononuclear phagocytes, but how it evades innate immune defenses of this indispensable primary innate immune cell is not well understood. This investigation revealed the molecular details of a ligand mimicry cellular reprogramming strategy that involved a short linear motif (SLiM), which enabled E. chaffeensis to exploit host cell signaling to establish and maintain infection. E. chaffeensis TRP120 is a moonlighting effector that has been associated with cellular activation and other functions, including ubiquitin ligase activity. Herein, we identified and demonstrated that a SLiM present within each tandem repeat of TRP120 activated Notch signaling. Notch is an evolutionarily conserved signaling pathway responsible for many cell functions, including cell fate, development, and innate immunity. This study is significant because it revealed the first molecularly defined pathogen encoded SLiM that appears to have evolved de novo to mimic endogenous Notch ligands. Understanding Notch activation during E. chaffeensis infection provides a model to study pathogen exploitation of signaling pathways and will be useful in developing molecularly targeted countermeasures for inhibiting infection by a multitude of disease-causing pathogens that exploit cell signaling through molecular mimicry.

Ehrlichia chaffeensis Outer Membrane Protein 1-Specific Human Antibody-Mediated Immunity Is Defined by Intracellular TRIM21-Dependent Innate Immune Activation and Extracellular Neutralization.

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.

Ehrlichia Activation of Wnt-PI3K-mTOR Signaling Inhibits Autolysosome Generation and Autophagic Destruction by the Mononuclear Phagocyte.

In multicellular organisms, autophagy is induced as an innate defense mechanism. Notably, the obligate intracellular bacterium Ehrlichia chaffeensis resides in early endosome-like vacuoles and circumvents lysosomal fusion through an unknown mechanism, thereby avoiding destruction in the autophagolysosome. In this report, we reveal that Wnt signaling plays a crucial role in inhibition of lysosomal fusion and autolysosomal destruction of ehrlichiae. During early infection, autophagosomes fuse with ehrlichial vacuoles to form an amphisome indicated by the presence of autophagy markers such as LC3 (microtubule-associated protein 1 light chain 3), Beclin-1, and p62. LC3 colocalized with ehrlichial morulae on days 1, 2, and 3 postinfection, and increased LC3II levels were detected during infection, reaching a maximal level on day 3. Ehrlichial vacuoles did not colocalize with the lysosomal marker LAMP2, and lysosomes were redistributed and dramatically reduced in level in the infected cells. An inhibitor specific for the Wnt receptor signaling component Dishevelled induced lysosomal fusion with ehrlichial inclusions corresponding to p62 degradation and promoted transcription factor EB (TFEB) nuclear localization. E. chaffeensis infection activated the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR (mechanistic target of rapamycin) pathway, and activation was induced by three ehrlichial tandem repeat protein (TRP) effectors, with TRP120 inducing the strongest activation. Moreover, induction of glycogen synthase kinase-3 (GSK3) performed using a Wnt inhibitor and small interfering RNA (siRNA) knockdown of critical components of PI3K-GSK3-mTOR signaling decreased ehrlichial survival. This report reveals Ehrlichia exploitation of the evolutionarily conserved Wnt pathway to inhibit autolysosome generation, thereby leading to evasion of this important innate immune defense mechanism.

Critical role of TLR4 in human metapneumovirus mediated innate immune responses and disease pathogenesis.

Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4⁻/⁻) were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4⁻/⁻ mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4⁻/⁻ mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.

Host-Viral Interactions: Role of Pattern Recognition Receptors (PRRs) in Human Pneumovirus Infections.

Acute respiratory tract infection (RTI) is a leading cause of morbidity and mortality worldwide and the majority of RTIs are caused by viruses, among which respiratory syncytial virus (RSV) and the closely related human metapneumovirus (hMPV) figure prominently. Host innate immune response has been implicated in recognition, protection and immune pathological mechanisms. Host-viral interactions are generally initiated via host recognition of pathogen-associated molecular patterns (PAMPs) of the virus. This recognition occurs through host pattern recognition receptors (PRRs) which are expressed on innate immune cells such as epithelial cells, dendritic cells, macrophages and neutrophils. Multiple PRR families, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs), contribute significantly to viral detection, leading to induction of cytokines, chemokines and type I interferons (IFNs), which subsequently facilitate the eradication of the virus. This review focuses on the current literature on RSV and hMPV infection and the role of PRRs in establishing/mediating the infection in both in vitro and in vivo models. A better understanding of the complex interplay between these two viruses and host PRRs might lead to efficient prophylactic and therapeutic treatments, as well as the development of adequate vaccines.