The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 (TANK-binding kinase 1) responds to microbial pathogens to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) (on S2159) to increase mTOR complex 1 (mTORC1) activity and signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Here we demonstrate that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation at physiological levels of protein expression. We find that TBK1 phosphorylates mTOR S2159 within mTORC2 in vitro, phosphorylates mTOR S2159 in cells, and interacts with mTORC2 in cells. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A), we show that TBK1 and mTOR S2159 phosphorylation increase mTORC2 catalytic activity and promote mTOR-dependent downstream signaling to Akt in response to several growth factors and poly(I:C). While microbial-derived stimuli activate TBK1, we find that growth factors fail to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, we propose that basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR complexes (mTORCs), key nodes within two major signaling systems. As TBK1 and mTORCs have each been linked to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.

Download Full-text

An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide

Molecules ◽

10.3390/molecules26247461 ◽

2021 ◽

Vol 26 (24) ◽

pp. 7461

Author(s):

Claire K. Holley ◽

Edward Cedrone ◽

Duncan Donohue ◽

Barry W. Neun ◽

Daniela Verthelyi ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Innate Immune Response ◽

Innate Immune ◽

Cytokine Secretion ◽

Poly I:C ◽

In Vitro Assays ◽

Innate Immune Responses ◽

Vitro Assay

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

Download Full-text

Combined Stimulation of Toll-Like Receptor 5 and NOD1 Strongly Potentiates Activity of NF-κB, Resulting in Enhanced Innate Immune Reactions and Resistance to Salmonella enterica Serovar Typhimurium Infection

Infection and Immunity ◽

10.1128/iai.00525-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3855-3864 ◽

Cited By ~ 23

Author(s):

Amir I. Tukhvatulin ◽

Ilya I. Gitlin ◽

Dmitry V. Shcheblyakov ◽

Natalia M. Artemicheva ◽

Lyudmila G. Burdelya ◽

...

Keyword(s):

Critical Role ◽

Immune Defense ◽

Innate Immune ◽

Microbial Infection ◽

Immune Reactions ◽

Content Type ◽

Essential Components ◽

Stimulation Of

ABSTRACTPathogen recognition receptors (PRRs) are essential components of host innate immune systems that detect specific conserved pathogen-associated molecular patterns (PAMPs) presented by microorganisms. Members of two families of PRRs, transmembrane Toll-like receptors (TLRs 1, 2, 4, 5, and 6) and cytosolic NOD receptors (NOD1 and NOD2), are stimulated upon recognition of various bacterial PAMPs. Such stimulation leads to induction of a number of immune defense reactions, mainly triggered via activation of the transcription factor NF-κB. While coordination of responses initiated via different PRRs sensing multiple PAMPS present during an infection makes clear biological sense for the host, such interactions have not been fully characterized. Here, we demonstrate that combined stimulation of NOD1 and TLR5 (as well as other NOD and TLR family members) strongly potentiates activity of NF-κB and induces enhanced levels of innate immune reactions (e.g., cytokine production) bothin vitroandin vivo. Moreover, we show that an increased level of NF-κB activity plays a critical role in formation of downstream responses. In live mice, synergy between these receptors resulting in potentiation of NF-κB activity was organ specific, being most prominent in the gastrointestinal tract. Coordinated activity of NOD1 and TLR5 significantly increased protection of mice against enteroinvasiveSalmonellainfection. Obtained results suggest that cooperation of NOD and TLR receptors is important for effective responses to microbial infectionin vivo.

Download Full-text

Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00512.2016 ◽

2018 ◽

Vol 315 (4) ◽

pp. F812-F823 ◽

Cited By ~ 7

Author(s):

Vijay Saxena ◽

David S. Hains ◽

John Ketz ◽

Melinda Chanley ◽

John D. Spencer ◽

...

Keyword(s):

Urinary Tract ◽

Mrna Expression ◽

Collecting Duct ◽

Immune Defense ◽

Innate Immune ◽

Targeted Analysis ◽

Collecting Tubule ◽

Tubule Cells

The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanism by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells that consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+mice to fluorescently label ICs and AQP2-cre+tdT+mice to fluorescently label PCs, and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure antimicrobial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC) challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge, this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.

Download Full-text

Detection of Microbial Infections Through Innate Immune Sensing of Nucleic Acids

Annual Review of Microbiology ◽

10.1146/annurev-micro-102215-095605 ◽

2018 ◽

Vol 72 (1) ◽

pp. 447-478 ◽

Cited By ~ 100

Author(s):

Xiaojun Tan ◽

Lijun Sun ◽

Jueqi Chen ◽

Zhijian J. Chen

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Immune Defense ◽

Innate Immune ◽

Type I Interferons ◽

Microbial Pathogens ◽

Type I ◽

Immune Recognition ◽

Microbial Infection ◽

Microbial Infections

Microbial infections are recognized by the innate immune system through germline-encoded pattern recognition receptors (PRRs). As most microbial pathogens contain DNA and/or RNA during their life cycle, nucleic acid sensing has evolved as an essential strategy for host innate immune defense. Pathogen-derived nucleic acids with distinct features are recognized by specific host PRRs localized in endolysosomes and the cytosol. Activation of these PRRs triggers signaling cascades that culminate in the production of type I interferons and proinflammatory cytokines, leading to induction of an antimicrobial state, activation of adaptive immunity, and eventual clearance of the infection. Here, we review recent progress in innate immune recognition of nucleic acids upon microbial infection, including pathways involving endosomal Toll-like receptors, cytosolic RNA sensors, and cytosolic DNA sensors. We also discuss the mechanisms by which infectious microbes counteract host nucleic acid sensing to evade immune surveillance.

Download Full-text

Enhancing innate antiviral immune responses in rainbow trout by double stranded RNA delivered with cationic phytoglycogen nanoparticles

Scientific Reports ◽

10.1038/s41598-019-49931-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Tamiru N. Alkie ◽

Jondavid de Jong ◽

Kristof Jenik ◽

Karl M. Klinger ◽

Stephanie J. DeWitte-Orr

Keyword(s):

Rainbow Trout ◽

Immune Responses ◽

Cell Line ◽

Culture Conditions ◽

Type I Interferons ◽

Poly I:C ◽

Synthetic Analogue ◽

Type I ◽

Viral Dsrna

Abstract Innate immunity is induced when pathogen-associated molecular patterns (PAMPs) bind host pattern recognition receptors (PRRs). Polyinosinic:polycytidylic acid [poly(I:C)] is a synthetic analogue of viral dsRNA that acts as a PAMP, inducing type I interferons (IFNs) in vertebrates. In the present study, the immunostimulatory effects of high molecular weight (HMW) poly(I:C) in rainbow trout cells were measured when bound to a cationic phytoglycogen nanoparticle (Nano-HMW). The physical characteristics of the nanoparticle itself, when bound to different lengths of dsRNA and when cell associated was evaluated. Optimal concentration and timing for innate immune stimulation was measured using the RTG-P1 reporter cell line. The immunostimulatory effects of HMW poly (I:C) was compared to Nano-HMW in vitro using the RTgutGC cell line cultured in a conventional monolayer or a transwell culture system. The ability of an activated intestinal epithelium to transmit an antiviral signal to macrophages was evaluated using a co-culture of RTgutGC cells and RTSll (a monocyte/macrophage cell). In all culture conditions, Nano-HMW was a more effective inducer of IFN-related antiviral immune responses compared to HMW poly (I:C) alone. This study introduces the use of cationic phytoglycogen nanoparticles as a novel delivery system for immunomodulatory molecules to enhance immune responses in aquatic vertebrates.

Download Full-text

Differential Regulation of β-Defensin Gene Expression during Cryptosporidium parvum Infection

Infection and Immunity ◽

10.1128/iai.72.5.2772-2779.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 2772-2779 ◽

Cited By ~ 78

Author(s):

Tarek K. Zaalouk ◽

Mona Bajaj-Elliott ◽

John T. George ◽

Vincent McDonald

Keyword(s):

Gene Expression ◽

Cryptosporidium Parvum ◽

Protozoan Parasite ◽

Immune Defense ◽

Microbial Pathogens ◽

In Vivo Models ◽

Defensin Gene ◽

Intestinal Epithelia

ABSTRACT Invasion of enterocytes by pathogenic microbes evokes both innate and adaptive immune responses, and microbial pathogens have developed strategies to overcome the initial host immune defense. β-Defensins are potentially important endogenous antibiotic-like effectors of innate immunity expressed by intestinal epithelia. In this study, the interplay between the enteric protozoan parasite Cryptosporidium parvum and host epithelial β-defensin expression was investigated. Using human and murine models of infection, we demonstrated that C. parvum infection differentially regulates β-defensin gene expression. Downregulation of murine β-defensin-1 mRNA and protein was observed in both in vitro and in vivo models of infection. Infection of the human colonic HT29 cell line with the parasite resulted in differential effects on various members of the defensin gene family. Partial reduction in human β-defensin-1 (hBD-1), induction of hBD-2, and no effect on hBD-3 gene expression was observed. Recombinant hBD-1 and hBD-2 peptides exhibited significant antimicrobial activity against C. parvum sporozoites in vitro. These findings demonstrate that C. parvum infection of enterocytes may affect the expression of various defensins in different ways and suggest that the overall outcome of the effect of antimicrobial peptides on early survival of the parasite may be complex.

Download Full-text

Indoleamine 2,3-Dioxygenase 1 Is a Lung-Specific Innate Immune Defense Mechanism That Inhibits Growth of Francisella tularensis Tryptophan Auxotrophs

Infection and Immunity ◽

10.1128/iai.00008-10 ◽

2010 ◽

Vol 78 (6) ◽

pp. 2723-2733 ◽

Cited By ~ 33

Author(s):

Kaitian Peng ◽

Denise M. Monack

Keyword(s):

Francisella Tularensis ◽

Defense Mechanisms ◽

Defense Mechanism ◽

Immune Defense ◽

The Body ◽

Innate Immune ◽

Microbial Pathogens ◽

Immune Mechanisms ◽

Indoleamine 2,3 Dioxygenase ◽

Organ Specific

ABSTRACT Upon microbial challenge, organs at various anatomic sites of the body employ different innate immune mechanisms to defend against potential infections. Accordingly, microbial pathogens evolved to subvert these organ-specific host immune mechanisms to survive and grow in infected organs. Francisella tularensis is a bacterium capable of infecting multiple organs and thus encounters a myriad of organ-specific defense mechanisms. This suggests that F. tularensis may possess specific factors that aid in evasion of these innate immune defenses. We carried out a microarray-based, negative-selection screen in an intranasal model of Francisella novicida infection to identify Francisella genes that contribute to bacterial growth specifically in the lungs of mice. Genes in the bacterial tryptophan biosynthetic pathway were identified as being important for F. novicida growth specifically in the lungs. In addition, a host tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase 1 (IDO1), is induced specifically in the lungs of mice infected with F. novicida or Streptococcus pneumoniae. Furthermore, the attenuation of F. novicida tryptophan mutant bacteria was rescued in the lungs of IDO1−/− mice. IDO1 is a lung-specific innate immune mechanism that controls pulmonary Francisella infections.

Download Full-text

TLR-induced reorganization of the IgM-BCR complex regulates B-1 cell responses to infections

10.1101/595694 ◽

2019 ◽

Author(s):

Hannah P. Savage ◽

Kathrin Kläsener ◽

Fauna L. Smith ◽

Zheng Luo ◽

Michael Reth ◽

...

Keyword(s):

Influenza Virus ◽

Salmonella Typhimurium ◽

Immune Defense ◽

Concomitant Increase ◽

Downstream Signaling ◽

Cell Responses ◽

Bcr Signaling ◽

Eventual Loss

AbstractNeonatally-developing, self-reactive B-1 cells generate steady levels natural antibodies throughout life. They can, however, also rapidly respond to infections with increased local antibody production. The mechanisms regulating these two seemingly very distinct functions are poorly understood, but have been linked to expression of CD5, an inhibitor of BCR-signaling. Here we demonstrate that TLR-mediated activation of CD5+ B-1 cells induced the rapid reorganization of the IgM-BCR complex, leading to the eventual loss of CD5 expression, and a concomitant increase in BCR-downstream signaling, both in vitro and in vivo after infections with influenza virus and Salmonella typhimurium. Both, initial CD5 expression and TLR-mediated stimulation, were required for the differentiation of B-1 cells to IgM-producing plasmablasts after infections. Thus, TLR-mediated signals support participation of B-1 cells in immune defense via BCR-complex reorganization.

Download Full-text

Innate Immune Responses to Wildtype and Attenuated Sheeppox Virus Mediated Through RIG-1 Sensing in PBMC In-Vitro

Frontiers in Immunology ◽

10.3389/fimmu.2021.666543 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tesfaye Rufael Chibssa ◽

Richard Thiga Kangethe ◽

Francisco J. Berguido ◽

Tirumala Bharani K. Settypalli ◽

Yang Liu ◽

...

Keyword(s):

Immune Responses ◽

Small Ruminants ◽

Significant Negative Correlation ◽

Individual Variability ◽

Innate Immune ◽

Economic Losses ◽

Viral Dsrna ◽

Sheeppox Virus

Sheeppox (SPP) is a highly contagious disease of small ruminants caused by sheeppox virus (SPPV) and predominantly occurs in Asia and Africa with significant economic losses. SPPV is genetically and immunologically closely related to goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which infect goats and cattle respectively. SPPV live attenuated vaccines (LAVs) are used for vaccination against SPP and goatpox (GTP). Mechanisms related to innate immunity elicited by SPPV are unknown. Although adaptive immunity is responsible for long-term immunity, it is the innate responses that prevent viral invasion and replication before LAVs generate specific long-term protection. We analyzed the relative expression of thirteen selected genes that included pattern recognition receptors (PRRs), Nuclear factor-κβ p65 (NF-κβ), and cytokines to understand better the interaction between SPPV and its host. The transcripts of targeted genes in sheep PBMC incubated with either wild type (WT) or LAV SPPV were analyzed using quantitative PCR. Among PRRs, we observed a significantly higher expression of RIG-1 in PBMC incubated with both WT and LAV, with the former producing the highest expression level. However, there was high inter-individual variability in cytokine transcripts levels among different donors, with the expression of TNFα, IL-15, and IL-10 all significantly higher in both PBMC infected with either WT or LAV compared to control PBMC. Correlation studies revealed a strong significant correlation between RIG-1 and IL-10, between TLR4, TNFα, and NF-κβ, between IL-18 and IL-15, and between NF-κβ and IL-10. There was also a significant negative correlation between RIG-1 and IFNγ, between TLR3 and IL-1 β, and between TLR4 and IL-15 (P< 0.05). This study identified RIG-1 as an important PRR in the signaling pathway of innate immune activation during SPPV infection, possibly through intermediate viral dsRNA. The role of immunomodulatory molecules produced by SPPV capable of inhibiting downstream signaling activation following RIG-1 upregulation is discussed. These findings advance our knowledge of the induction of immune responses by SPPV and will help develop safer and more potent vaccines against SPP and GTP.

Download Full-text

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

Journal of Experimental Medicine ◽

10.1084/jem.20100257 ◽

2010 ◽

Vol 207 (8) ◽

pp. 1745-1755 ◽

Cited By ~ 347

Author(s):

Petr Broz ◽

Kim Newton ◽

Mohamed Lamkanfi ◽

Sanjeev Mariathasan ◽

Vishva M. Dixit ◽

...

Keyword(s):

Salmonella Typhimurium ◽

Host Defense ◽

Immune Defense ◽

Innate Immune ◽

Microbial Pathogens ◽

Intracellular Pathogens ◽

Danger Signals ◽

Caspase 1 ◽

Innate Immune Defense

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro–IL-1β processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

Download Full-text