Self-Complementary AAV Vectors Cause a Substantially Heightened TLR9-Dependent Innate Immune Response In the Liver

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 252-252 ◽  
Author(s):  
Ashley T Martino ◽  
Masataka Suzuki ◽  
David Markusic ◽  
Irene Zolotukhin ◽  
Daniel A Muruve ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Immune Responses ◽  
Fold Increase ◽  
Innate Immune ◽  
High Dose ◽  
Wild Type ◽  
Innate Response ◽  
Tnf Α ◽  
Capsid Sequence

Abstract Abstract 252 Successful gene therapy for hemophilia requires therapeutic expression in the absence of destructive immune responses. Clinical trials have revealed that adaptive immune responses eliminated therapeutic levels of factor IX (F.IX) following high-dose hepatic in vivo gene transfer using single stranded (ss) AAV serotype 2 vector. More efficient AAV vectors have been developed using AAV2 with Y-F capsid mutations, alternate serotypes such as AAV8, and self-complimentary (sc) rAAV vectors. Innate immune responses to AAV in the liver are thought to be weak and highly transient. Our study sought to investigate whether variations in the capsid or genome will alter the immune profile. AAV vectors were delivered into the portal circulation of C3H/OuJ mice at 1011 vector genomes per mouse. We tested ssAAV-2, ssAAV2-triple Y-F mutant, ssAAV-8 vectors as well as scAAV-2 and scAAV-8 vectors, all expressing the hF.IX transgene. Quantitative RT-PCR array showed a mild transient up-regulation of MyD88, TLR-9, TNF-α, MCP-1, IP-10 and IFN-α/β within 2 hrs, which subsided by 6 hrs following delivery of ssAAV vectors, regardless of the capsid sequence/efficiency of hepatocyte transduction. In contrast, scAAV-2 and scAAV-8 vectors induced 4- to 8-fold increases in TLR-2, TLR-9, MyD88, TNF-α, MCP-1, IP-10, and IFN-α/β expression when compared to the ssAAV vectors. In addition, scAAV vectors induced expression of pro-inflammatory cytokines and chemokines not detected for ssAAV: IL-6, IL-12α, MIP-1, and RANTES. Paralleling this data was a 4-fold increase in local protein levels of IL-6, TNF-α, and MCP-1, and a systemic increase in IL-6. None of the vectors activated TLR-4, indicating that these effects cannot be attributed to LPS contamination. Several other innate response genes showed no differences in expression compared to PBS injected mice, which included IL-1α, IL-1β, KC, TLR-1, and TLR-3 to -8. The heightened innate response to scAAV vectors was further characterized by a 5-fold increase in macrophage and neutrophil infiltrates in liver sections, illustrating the potential for hepatotoxicity. Innate responses were absent in TLR-9 deficient C57BL/6 mice and in wild-type mice that had received an oligonucleotide ligand inhibitory to TLR-9. Compared to wild-type mice, hFIX levels were increased 2-fold in TLR-9 deficient mice following scAAV vector delivery and antibody formation against capsid was delayed. Additionally, in vitro studies with a TLR-9 reporter cell line showed significantly increased TLR-9 signaling for scAAV2 compared to ssAAV vectors, independent of the transgene/expression cassette. Neither vector activated the inflammasome. Further in vivo studies showed that the innate response to scAAV was, with the exception of TNF-a expression, Kupffer cell dependent, likely owing to the importance of these cells to sequester viral particles in the liver. Our results have several implications. Gene therapy applications based on administration of high-dose scAAV may be limited by immunotoxicities, which, however, can be prevented by inhibition of TLR-9 signaling. Of general importance, changing the genome configuration of a virus from single- to double-stranded DNA increased sensing by the endosomal receptor TLR-9, thereby enhancing innate immunity. Disclosures: Herzog: Genzyme Corp: Patents & Royalties.

The atypical chemokine receptor CCX-CKR scavenges homeostatic chemokines in circulation and tissues and suppresses Th17 responses

Blood ◽  
2010 ◽  
Vol 116 (20) ◽  
pp. 4130-4140 ◽  
Author(s):  
Iain Comerford ◽  
Robert J. B. Nibbs ◽  
Wendel Litchfield ◽  
Mark Bunting ◽  
Yuka Harata-Lee ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Lymph Nodes ◽  
Chemokine Receptor ◽  
Fold Increase ◽  
Wild Type ◽  
Peripheral Lymph ◽  
The Central Nervous System

Abstract Our previous in vitro studies led to proposals that the atypical chemokine receptor CCX-CKR is a scavenger of CCR7 ligand homeostatic chemokines. In the present study, we generated CCX-CKR−/− mice and confirm this scavenger function in vivo. Compared with wild-type mice, CCX-CKR−/− have a 5-fold increase in the level of CCL21 protein in blood, and 2- to 3-fold increases in CCL19 and CCL21 in peripheral lymph nodes. The effect of these protein increases on immunity was investigated after immunization with MOG35-55 peptide emulsified in complete Freund adjuvant (CFA). The subsequent characteristic paralysis develops with enhanced kinetics and severity in CCX-CKR−/− versus wild-type mice. Despite this effect, antigen-specific immune responses in the draining lymph nodes are diminished in CCX-CKR−/− mice. Instead, the earlier onset of disease is associated with enhanced T-cell priming in the CCX-CKR−/− spleen and a skewing of CD4+ T-cell responses toward Th17 rather than Th1. This observation correlates with increased expression of IL-23 in the CCX-CKR−/− spleen and increased CCL21 levels in the central nervous system postimmunization. The early onset of disease in CCX-CKR−/− mice is reversed by systemic administration of neutralizing anti-CCL21 antibodies. Thus, by regulating homeostatic chemokine bioavailability, CCX-CKR influences the localization, kinetics, and nature of adaptive immune responses in vivo.

scholarly journals Viable Borrelia burgdorferi Enhances Interleukin-10 Production and Suppresses Activation of Murine Macrophages

2007 ◽  
Vol 76 (3) ◽  
pp. 1153-1162 ◽  
Author(s):  
John J. Lazarus ◽  
Maria A. Kay ◽  
Akisha L. McCarter ◽  
R. Mark Wooten
Keyword(s):  
Borrelia Burgdorferi ◽  
Immune Responses ◽  
Culture Medium ◽  
Interleukin 10 ◽  
Innate Immune ◽  
Innate Immune Responses ◽  
Wild Type ◽  
Adaptive Immune

ABSTRACT Although it is capable of eliciting strong innate and adaptive immune responses, Borrelia burgdorferi often evades immune clearance through largely unknown mechanisms. Our previous studies determined that infected interlukin-10−/− (IL-10−/−) mice show significantly lower B. burgdorferi levels than wild-type (B6) mice and that IL-10 inhibits innate immune responses critical for controlling B. burgdorferi infection. To determine whether virulent B. burgdorferi preferentially enhances IL-10 production, we developed an in vitro coculture medium (RPMI.B) in which both B. burgdorferi and primary macrophages (Mφs) remain viable. B. burgdorferi grew at similar rates and was able to regulate expression of immunoreactive proteins with similar kinetics in RPMI.B and in traditional BSK medium; in contrast, B. burgdorferi cultured in conventional tissue culture medium (RPMI) rapidly lost viability. Coculture of viable B. burgdorferi in RPMI.B with Mφs resulted in more rapid and significant increases in IL-10 transcripts and secreted proteins than coculture with nonviable B. burgdorferi in RPMI, which corresponded with decreased production of proinflammatory cytokines. Addition of live B. burgdorferi to Mφs in RPMI.B also elicited substantially higher IL-10 levels than heat-killed bacteria elicited, confirming that increased IL-10 production was not inherent to coculture in RPMI.B. Transfer of supernatants from B. burgdorferi-stimulated Mφs into naïve Mφ cultures resulted in suppressed activation upon subsequent stimulation with different bacterial agonists, and this suppression was obviated by IL-10-specific antibody. In vivo analyses determined that murine skin samples exhibited substantial upregulation of IL-10 within 24 h of injection of B. burgdorferi. Together, these results suggest that viable B. burgdorferi can suppress early Mφ responses during infection by causing increased release of IL-10.

scholarly journals Regulation of Pulmonary and Systemic Bacterial Lipopolysaccharide Responses in Transgenic Mice Expressing Human Elafin

2003 ◽  
Vol 71 (7) ◽  
pp. 3766-3774 ◽  
Author(s):  
J.-M. Sallenave ◽  
G. A. Cunningham ◽  
R. M. James ◽  
G. McLachlan ◽  
C. Haslett
Keyword(s):  
Immune Responses ◽  
Inflammatory Responses ◽  
Innate Immune ◽  
Bacterial Lipopolysaccharide ◽  
Dual Function ◽  
Innate Immune Responses ◽  
Wild Type ◽  
Necrosis Factor Alpha ◽  
Elastase Inhibitor ◽  
Tnf Α

ABSTRACT The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-α compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation.

scholarly journals Role of TLR4 in Persistent Leptospira interrogans Infection: A Comparative In Vivo Study in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Nisha Nair ◽  
Mariana S. Guedes ◽  
Adeline M. Hajjar ◽  
Catherine Werts ◽  
Maria Gomes-Solecki
Keyword(s):  
Innate Immune ◽  
High Dose ◽  
Wild Type ◽  
Immune Markers ◽  
Lethal Infection ◽  
Tlr4 Gene ◽  
Kinetics Of ◽  
Overall Kinetics

Toll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. We infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wild-type (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Thus, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis.

scholarly journals MyD88-Dependent Signaling Affects the Development of Meningococcal Sepsis by Nonlipooligosaccharide Ligands

2006 ◽  
Vol 74 (6) ◽  
pp. 3538-3546 ◽  
Author(s):  
Laura Plant ◽  
Hong Wan ◽  
Ann-Beth Jonsson
Keyword(s):  
Inflammatory Responses ◽  
Innate Immune ◽  
Meningococcal Sepsis ◽  
Wild Type ◽  
Microbial Infections ◽  
In The Wild ◽  
Myeloid Differentiation Factor ◽  
Dependent Pathway ◽  
Meningococcal Strain

ABSTRACT The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88−/− mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88−/− mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

Effect of aerobic exercise on innate immune responses and inflammatory mediators in the spinal cord of diabetic rats

2020 ◽  
Vol 16 (4) ◽  
pp. 293-301
Author(s):  
A. Kaki ◽  
M. Nikbakht ◽  
A.H. Habibi ◽  
H.F. Moghadam
Keyword(s):  
Spinal Cord ◽  
Neuropathic Pain ◽  
Immune Responses ◽  
Diabetic Neuropathy ◽  
Aerobic Exercise ◽  
Inflammatory Mediators ◽  
Innate Immune ◽  
Moderate Intensity ◽  
Innate Immune Responses ◽  
Tnf Α

Neuronal inflammation is one of the pathophysiological causes of diabetes neuropathic pain. The purpose of this research was to determine the effect of aerobic exercise on innate immune responses and inflammatory mediators in the spinal dorsal horn in rats with diabetic neuropathic pain. 40 eight-week-old male Wistar rats (weight range 220±10.2 g) were randomly divided into four groups of (1) sedentary diabetic neuropathy (SDN), (2) training diabetic neuropathy (TDN), (3) training control (TC), and (4) sedentary control (SC). Diabetes was induced by injection of streptozocin (50 mg/kg). Following confirmation of behavioural tests for diabetes neuropathy, the training groups performed 6 weeks of moderate-intensity aerobic exercise on the treadmill. The expression of Toll like receptor (TLR)4, TLR2, tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-10 genes in L4-L6 spinal cord sensory neurons was measured by Real Time PCR. Two-way ANOVA and Bonferroni’s post hoc tests were used for statistical analysis. After performing aerobic exercise protocol, the TDN compared to the SDN showed a significant decrease in the mean score of pain in the formalin test and a significant increase in the latency in Tail-Flick test was observed. The expression of TLR4, TLR2, TNF-α and IL-1β genes was significantly higher in the SDN than in the SC group (P<0.05). The expression of the above genes in the TDN was significantly lower than the SDN group (P<0.05). Also, the expression level of IL-10 gene was significantly higher in the TDN than the SDN group (P<0.05). Aerobic exercise improved sensitivity of nociceptors to pain-inducing agents in diabetic neuropathy due to inhibition of inflammatory receptors and increased levels of anti-inflammatory agents in the nervous system. Thus, aerobic exercise should be used as a non-pharmacological intervention for diabetic patients to reduce neuropathic pain.

scholarly journals Occurrence of foamy macrophages during the innate response of zebrafish to trypanosome infections

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sem H Jacobs ◽  
Eva Dóró ◽  
Ffion R Hammond ◽  
Mai E Nguyen-Chi ◽  
Georges Lutfalla ◽  
...  
Keyword(s):  
Immune Response ◽  
Early Response ◽  
Innate Immune ◽  
Infection Model ◽  
Parasite Control ◽  
Innate Response ◽  
Larval Zebrafish ◽  
Inflammatory Profile ◽  
Foamy Macrophages

A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In this study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterize their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the occurrence of foamy macrophages characterized by a strong pro-inflammatory profile and potentially associated with an exacerbated immune response as well as susceptibility to the infection. To our knowledge this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.

IL-4 increases surfactant and regulates metabolism in vivo

2000 ◽  
Vol 278 (1) ◽  
pp. L75-L80 ◽  
Author(s):  
Machiko Ikegami ◽  
Jeffrey A. Whitsett ◽  
Zissis C. Chroneos ◽  
Gary F. Ross ◽  
Jacquelyn A. Reed ◽  
...  
Keyword(s):  
Lipid Synthesis ◽  
Fold Increase ◽  
Surfactant Protein ◽  
Clara Cells ◽  
Wild Type ◽  
Surfactant Lipid ◽  
Alveolar Proteinosis ◽  
Selective Increase

Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4.8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.

scholarly journals Toll-like Receptor 7 Contributes to Inflammation, Organ Injury, and Mortality in Murine Sepsis

2019 ◽  
Vol 131 (1) ◽  
pp. 105-118 ◽  
Author(s):  
Wenling Jian ◽  
Lili Gu ◽  
Brittney Williams ◽  
Yan Feng ◽  
Wei Chao ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Immune Responses ◽  
Knockout Mice ◽  
Cecal Ligation ◽  
Kidney Injury ◽  
Innate Immune ◽  
Organ Injury ◽  
Innate Immune Responses ◽  
Toll Like Receptor ◽  
Wild Type

Abstract Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Sepsis remains a critical illness with high mortality. The authors have recently reported that mouse plasma RNA concentrations are markedly increased during sepsis and closely associated with its severity. Toll-like receptor 7, originally identified as the sensor for single-stranded RNA virus, also mediates host extracellular RNA-induced innate immune responses in vitro and in vivo. Here, the authors hypothesize that innate immune signaling via Toll-like receptor 7 contributes to inflammatory response, organ injury, and mortality during polymicrobial sepsis. Methods Sepsis was created by (1) cecal ligation and puncture or (2) stool slurry peritoneal injection. Wild-type and Toll-like receptor 7 knockout mice, both in C57BL/6J background, were used. The following endpoints were measured: mortality, acute kidney injury biomarkers, plasma and peritoneal cytokines, blood bacterial loading, peritoneal leukocyte counts, and neutrophil phagocytic function. Results The 11-day overall mortality was 81% in wild-type mice and 48% in Toll-like receptor 7 knockout mice after cecal ligation and puncture (N = 27 per group, P = 0.0031). Compared with wild-type septic mice, Toll-like receptor 7 knockout septic mice also had lower sepsis severity, attenuated plasma cytokine storm (wild-type vs. Toll-like receptor 7 knockout, interleukin-6: 43.2 [24.5, 162.7] vs. 4.4 [3.1, 12.0] ng/ml, P = 0.003) and peritoneal inflammation, alleviated acute kidney injury (wild-type vs. Toll-like receptor 7 knockout, neutrophil gelatinase-associated lipocalin: 307 ± 184 vs.139 ± 41-fold, P = 0.0364; kidney injury molecule-1: 40 [16, 49] vs.13 [4, 223]-fold, P = 0.0704), lower bacterial loading, and enhanced leukocyte peritoneal recruitment and phagocytic activities at 24 h. Moreover, stool slurry from wild-type and Toll-like receptor 7 knockout mice resulted in similar level of sepsis severity, peritoneal cytokines, and leukocyte recruitment in wild-type animals after peritoneal injection. Conclusions Toll-like receptor 7 plays an important role in the pathogenesis of polymicrobial sepsis by mediating host innate immune responses and contributes to acute kidney injury and mortality.

scholarly journals MGL1 Receptor Plays a Key Role in the Control of T. cruzi Infection by Increasing Macrophage Activation through Modulation of ERK1/2, c-Jun, NF-κB and NLRP3 Pathways

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 108
Author(s):  
Tonathiu Rodriguez ◽  
Thalia Pacheco-Fernández ◽  
Alicia Vázquez-Mendoza ◽  
Oscar Nieto-Yañez ◽  
Imelda Juárez-Avelar ◽  
...  
Keyword(s):  
Immune Responses ◽  
Macrophage Activation ◽  
Cell Activation ◽  
Wild Type ◽  
Mhc Ii ◽  
Adaptive Immune ◽  
Splenic Cell ◽  
Tnf Α ◽  
Cruzi Infection

Macrophage galactose-C type lectin (MGL)1 receptor is involved in the recognition of Trypanosoma cruzi (T. cruzi) parasites and is important for the modulation of the innate and adaptive immune responses. However, the mechanism by which MGL1 promotes resistance to T. cruzi remains unclear. Here, we show that MGL1 knockout macrophages (MGL1−/− Mφ) infected in vitro with T. cruzi were heavily parasitized and showed decreased levels of reactive oxygen species (ROS), nitric oxide (NO), IL-12 and TNF-α compared to wild-type macrophages (WT Mφ). MGL1−/− Mφ stimulated in vitro with T. cruzi antigen (TcAg) showed low expression of TLR-2, TLR-4 and MHC-II, which resulted in deficient splenic cell activation compared with similar co-cultured WT Mφ. Importantly, the activation of p-ERK1/2, p-c-Jun and p-NF-κB p65 were significantly reduced in MGL1−/− Mφ exposed to TcAg. Similarly, procaspase 1, caspase 1 and NLRP3 inflammasome also displayed a reduced expression that was associated with low IL-β production. Our data reveal a previously unappreciated role for MGL1 in Mφ activation through the modulation of ERK1/2, c-Jun, NF-κB and NLRP3 signaling pathways, and to the development of protective innate immunity against experimental T. cruzi infection.

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