Chronic HIV Infection Increases Monocyte NLRP3 Inflammasome-Dependent IL-1α and IL-1ß Release.

Antiretroviral treatment (ART) has converted HIV from a lethal disease to a chronic condition, yet co-morbidities persist. Incomplete immune recovery and chronic immune activation, especially in the gut mucosa, contribute to these complications. Inflammasomes, multi-protein complexes activated by innate immune receptors, appear to play a role in these inflammatory responses. In particular, preliminary data indicate the involvement of IFI16 and NLRP3 inflammasomes in chronic HIV infection. This study explores inflammasome function in monocytes from people with HIV (PWH); 22 ART-treated with suppressed viremia and 17 untreated PWH were compared to 33 HIV-negative donors. Monocytes were primed with LPS and inflammasomes activated with ATP in vitro. IFI16 and NLRP3 mRNA expression were examined in a subset of donors. IFI16 and NLRP3 expression in unstimulated monocytes correlated negatively with CD4 T cell counts in untreated PWH. For IFI16, there was also a positive correlation with viral load. Monocytes from untreated PWH exhibit increased release of IL-1α, IL-1ß, and TNF compared to treated PWH and HIV-negative donors. However, circulating monocytes in PWH are not pre-primed for inflammasome activation in vivo. The findings suggest a link between IFI16, NLRP3, and HIV progression, emphasizing their potential role in comorbidities such as cardiovascular disease. The study provides insights into inflammasome regulation in HIV pathogenesis and its implications for therapeutic interventions.

CD38 deficient mice are not protected from atherosclerosis.

CD38 is a multifunctional enzyme implicated in chemotaxis of myeloid cells and lymphocyte activation, but also expressed by resident cells such as endothelial and smooth muscle cells. CD38 is important for host defense against microbes. However, CD38's role in the pathogenesis of atherosclerosis is controversial with seemingly conflicting results reported so far. To clarify the discrepancy of current literature on the effect of CD38 ablation on atherosclerosis development, we implanted a shear stress modifier around the right carotid artery in CD38-/- and WT mice. Hypercholesterolemia was induced by human gain-of-function PCSK9 (D374Y), introduced using AAV vector (serotype 9), combined with an atherogenic diet for a total of 9 weeks. Atherosclerosis was assessed at the aortic root, aortic arch and the right carotid artery. The findings can be summarized as follows: i) CD38-/- and WT mice had a similar atherosclerotic burden in all three locations, ii) No significant differences in monocyte infiltration or macrophage content could be seen in the plaques, and iii) The amount of collagen deposition in the plaques were also similar between CD38-/- and WT mice. In conclusion, our data suggest that CD38-/- mice are neither protected against nor prone to atherosclerosis compared to WT mice.

Clinical consequences of infliximab immunogenicity and the effect of proactive therapeutic drug monitoring: exploratory analyses of the randomised, controlled NOR-DRUM trials.

BACKGROUND: Antidrug antibodies to TNF inhibitors might affect clinical outcomes. Proactive therapeutic drug monitoring allows for early detection of antidrug antibodies and might reduce negative clinical consequences. We aimed to explore how antidrug antibodies to the TNF inhibitor infliximab influence treatment outcomes, and to assess the effect of proactive therapeutic drug monitoring. METHODS: This was a predefined exploratory analysis of data from the randomised, controlled NOR-DRUM trials. The trials were conducted in rheumatology, gastroenterology, and dermatology departments at 21 Norwegian hospitals. Adult patients (aged 18-75 years) with immune-mediated inflammatory diseases were randomly assigned to proactive therapeutic drug monitoring or standard infliximab dosing in the NOR-DRUM A trial (30-week follow-up) and the NOR-DRUM B trial (52-week follow-up). Antidrug antibodies were assessed with a drug-sensitive assay before each infusion. The outcomes of remission (at week 30), disease worsening (during 52 weeks), infusion reactions, and infliximab discontinuation were assessed according to the presence of antidrug antibodies and use of therapeutic drug monitoring. FINDINGS: Between March 1, 2017, and Dec 12, 2019, 616 patients were included in the NOR-DRUM trials, of whom 615 had at least one serum infliximab and antidrug antibody assessment and were included in the present analyses. Mean age was 45 years (IQR 32-56), 305 (50%) patients were women, and 310 (50%) patients were men. Antidrug antibodies were detected in 147 (24%) patients. Remission at week 30 occurred in 25 (35%) of 72 patients with antidrug antibodies and 180 (54%) of 335 without antidrug antibodies (risk ratio 0·62 [95% CI 0·45-0·86]; p=0·0037). In patients with antidrug antibodies compared with patients without antidrug antibodies, higher rates were found for: disease worsening over 52 weeks (0·76 per person-year vs 0· 35 per person-year, hazard ratio [HR] 2·02 [95% CI 1·33-3·07]; p=0·0009), infusion reactions (0·16 per person-year vs 0·03 per person-year, HR 17·02 [6·98-41·47]; p<0·0001), and infliximab discontinuation (1·00 per person-year vs 0·20 per person-year, HR 6·64 [4·84-9·11]; p<0·0001). These associations were more pronounced in patients with high concentrations of antidrug antibodies than in those with low concentrations of antidrug antibodies. Independent of antibody status, therapeutic drug monitoring was associated with a lower risk of disease worsening (HR 0·41 [0·29-0·59]; p=0·0001) or an infusion reaction (HR 0·30 [0·12-0·73]; p=0·0076), and was associated with an increase in the rate of infliximab discontinuation (HR 1·37 [1·02-1·83]; p=0·037). INTERPRETATION: In patients where antidrug antibodies were detected, remission was less likely to be reached and sustained, and infusion reaction or discontinuation of infliximab was more likely. Timely detection of antidrug antibodies by proactive therapeutic drug monitoring facilitated treatment decisions that reduced the negative consequences, both regarding infliximab effectiveness and safety. This highlights the role of proactive therapeutic drug monitoring in optimising infliximab therapy. FUNDING: Inter-regional KLINBEFORSK grants and South-Eastern Norway Regional Health Authority grants.

HLA-DQ2 is associated with anti-drug antibody formation to infliximab in patients with immune-mediated inflammatory diseases.

BACKGROUND: Immunogenicity to tumour necrosis factor inhibitors is a significant clinical problem leading to treatment failure and adverse events. The study aimed to assess human leukocyte antigen (HLA) associations with anti-drug antibody (ADAb) formation to infliximab. METHODS: Immune-mediated inflammatory disease patients on infliximab therapy (n = 612) were included. Neutralising ADAb were assessed with a drug-sensitive assay. Next generation sequencing-based HLA typing was performed. RESULTS: Overall, 147 (24%) patients developed ADAb. Conditional analyses indicated HLA-DQB1 (p = 1.4 × 10-6 ) as a primary risk locus. Highest risk of ADAb was seen when carrying at least one of the HLA-DQ2 haplotypes; DQB1*02:01-DQA1*05:01 or DQB1*02:02-DQA1*02:01 (OR 3.18, 95% CI 2.15-4.69 and p = 5.9 × 10-9 ). Results were consistent across diseases and when adjusting for concomitant immunomodulator. Computational predictions indicated that these HLA-DQ2 haplotypes bind to peptide motifs from infliximab light chain. CONCLUSION: A genome-wide significant association between two HLA-DQ2 haplotypes and the risk of ADAb formation to infliximab was identified, suggesting that HLA-DQ2 testing may facilitate personalised treatment decisions.

Risk factors for anti-drug antibody formation to infliximab: Secondary analyses of a randomised controlled trial.

BACKGROUND: Anti-drug antibodies (ADAb) frequently form early in the treatment course of infliximab and other tumour necrosis factor (TNF) inhibitors, leading to treatment failure and adverse events. OBJECTIVE: To identify risk factors for ADAb in the early phase of infliximab treatment. METHODS: Patients (n = 410) with immune-mediated inflammatory diseases who initiated infliximab treatment were included in the 38-week Norwegian Drug Monitoring Trial (NOR-DRUM) A and randomised 1:1 to therapeutic drug monitoring (TDM) or standard therapy. Serum levels of infliximab and ADAb were measured at each infusion. Possible risk factors for ADAb formation were assessed using logistic regression, adjusting for potential confounders. RESULTS: ADAb were detected in 78 (19%) patients. A diagnosis of rheumatoid arthritis (RA) (odds ratio [OR], 1.9 [95% confidence interval [CI] 1.0-3.6]) and lifetime smoking (OR, 2.0 [CI 1.1-3.6]) were baseline risk factors, while baseline use of concomitant immunosuppressors (OR, 0.4 [CI 0.2-0.8]) and a diagnosis of spondyloarthritis (SpA) (OR, 0.4 [CI 0.2-0.8]) reduced the risk of ADAb. Higher disease activity during follow-up (OR, 1.1 [CI 1.0-1.1]) and "drug holidays" of more than 11 weeks (OR, 4.1 [CI 1.2-13.8]) increased the risk of ADAb, whereas higher infliximab doses (OR, 0.1 [CI 0.0-0.3) and higher serum infliximab concentrations (OR, 0.7 [CI 0.6-0.8]) reduced the risk of immunogenicity. CONCLUSION: Several risk factors for ADAb formation during early-phase infliximab treatment were identified. This knowledge provides a basis for treatment strategies to mitigate the formation of ADAb and identify patients in whom these measures are of particular importance.

Targeting the Inflammasome in Cardiovascular Disease.

The pathogenesis of cardiovascular disease (CVD) is complex and multifactorial, and inflammation plays a central role. Inflammasomes are multimeric protein complexes that are activated in a 2-step manner in response to infection or tissue damage. Upon activation the proinflammatory cytokines, interleukins-1ß and -18 are released. In the last decade, the evidence that inflammasome activation plays an important role in CVD development became stronger. We discuss the role of different inflammasomes in the pathogenesis of CVD, focusing on atherosclerosis and heart failure. This review also provides an overview of existing experimental studies and clinical trials on inflammasome inhibition as a therapeutic target in these disorders.

Effect of Therapeutic Drug Monitoring vs Standard Therapy During Maintenance Infliximab Therapy on Disease Control in Patients With Immune-Mediated Inflammatory Diseases: A Randomized Clinical Trial.

Importance: Proactive therapeutic drug monitoring (TDM), consisting of individualized treatment based on scheduled assessments of serum drug levels, has been proposed as an alternative to standard therapy to optimize efficacy and safety of infliximab and other biologic drugs. However, it remains unclear whether proactive TDM improves clinical outcomes during maintenance therapy. Objective: To assess whether proactive TDM during maintenance therapy with infliximab improves treatment efficacy by preventing disease worsening compared with standard infliximab therapy without TDM. Design, Setting, and Participants: Randomized, parallel-group, open-label clinical trial including 458 adults with rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, ulcerative colitis, Crohn disease, or psoriasis undergoing maintenance therapy with infliximab in 20 Norwegian hospitals. Patients were recruited from June 7, 2017, to December 12, 2019. Final follow-up took place on December 14, 2020. Interventions: Patients were randomized 1:1 to proactive TDM with dose and interval adjustments based on scheduled monitoring of serum drug levels and antidrug antibodies (TDM group; n = 228) or to standard infliximab therapy without drug and antibody level monitoring (standard therapy group; n = 230). Main Outcome and Measures: The primary outcome was sustained disease control without disease worsening, defined by disease-specific composite scores or consensus about disease worsening between patient and physician leading to a major change in treatment (switching to another biologic drug, adding an immunosuppressive drug including glucocorticoids, or increasing the infliximab dose), during the 52-week study period. Results: Among 458 randomized patients (mean age, 44.8 [SD, 14.3] years; 216 women [49.8%]), 454 received their randomly allocated intervention and were included in the full analysis set. The primary outcome of sustained disease control without disease worsening was observed in 167 patients (73.6%) in the TDM group and 127 patients (55.9%) in the standard therapy group. The estimated adjusted difference was 17.6% (95% CI, 9.0%-26.2%; P < .001) favoring TDM. Adverse events were reported in 137 patients (60%) and 142 patients (63%) in the TDM and standard therapy groups, respectively. Conclusions and Relevance: Among patients with immune-mediated inflammatory diseases undergoing maintenance therapy with infliximab, proactive TDM was more effective than treatment without TDM in sustaining disease control without disease worsening. Further research is needed to compare proactive TDM with reactive TDM, to assess the effects on long-term disease complications, and to evaluate the cost-effectiveness of this approach. Trial Registration: ClinicalTrials.gov Identifier: NCT03074656.

Effect of Therapeutic Drug Monitoring vs Standard Therapy During Infliximab Induction on Disease Remission in Patients With Chronic Immune-Mediated Inflammatory Diseases: A Randomized Clinical Trial.

Importance: Proactive therapeutic drug monitoring (TDM), defined as individualized drug dosing based on scheduled monitoring of serum drug levels, has been proposed as an alternative to standard therapy to maximize efficacy and safety of infliximab and other biological drugs. However, whether proactive TDM improves clinical outcomes when implemented at the time of drug initiation, compared with standard therapy, remains unclear. Objective: To assess whether TDM during initiation of infliximab therapy improves treatment efficacy compared with standard infliximab therapy without TDM. Design, Setting, and Participants: Randomized, parallel-group, open-label clinical trial of 411 adults with rheumatoid arthritis, spondyloarthritis, psoriatic arthritis, ulcerative colitis, Crohn disease, or psoriasis initiating infliximab therapy in 21 hospitals in Norway. Patients were recruited from March 1, 2017, to January 10, 2019. Final follow-up occurred on November 5, 2019. Interventions: Patients were randomized 1:1 to receive proactive TDM with dose and interval adjustments based on scheduled monitoring of serum drug levels and antidrug antibodies (TDM group; n = 207) or standard infliximab therapy without drug and antibody level monitoring (standard therapy group; n = 204). Main Outcomes and Measures: The primary end point was clinical remission at week 30. Results: Among 411 randomized patients (mean age, 44.7 [SD, 14.9] years; 209 women [51%]), 398 (198 in the TDM group and 200 in the standard therapy group) received their randomized intervention and were included in the full analysis set. Clinical remission at week 30 was achieved in 100 (50.5%) of 198 and 106 (53.0%) of 200 patients in the TDM and standard therapy groups, respectively (adjusted difference, 1.5%; 95% CI, -8.2% to 11.1%; P = .78). Adverse events were reported in 135 patients (68%) and 139 patients (70%) in the TDM and standard therapy groups, respectively. Conclusions and Relevance: Among patients with immune-mediated inflammatory diseases initiating treatment with infliximab, proactive therapeutic drug monitoring, compared with standard therapy, did not significantly improve clinical remission rates over 30 weeks. These findings do not support routine use of therapeutic drug monitoring during infliximab induction for improving disease remission rates. Trial Registration: ClinicalTrials.gov Identifier: NCT03074656.

Cholesterol crystals use complement to increase NLRP3 signaling pathways in coronary and carotid atherosclerosis.

BACKGROUND: During atherogenesis, cholesterol precipitates into cholesterol crystals (CC) in the vessel wall, which trigger plaque inflammation by activating the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. We investigated the relationship between CC, complement and NLRP3 in patients with cardiovascular disease. METHODS: We analysed plasma, peripheral blood mononuclear cells (PBMC) and carotid plaques from patients with advanced atherosclerosis applying ELISAs, multiplex cytokine assay, qPCR, immunohistochemistry, and gene profiling. FINDINGS: Transcripts of interleukin (IL)-1beta(ß) and NLRP3 were increased and correlated in PBMC from patients with acute coronary syndrome (ACS). Priming of these cells with complement factor 5a (C5a) and tumour necrosis factor (TNF) before incubation with CC resulted in increased IL-1ß protein when compared to healthy controls. As opposed to healthy controls, systemic complement was significantly increased in patients with stable angina pectoris or ACS. In carotid plaques, complement C1q and C5b-9 complex accumulated around CC-clefts, and complement receptors C5aR1, C5aR2 and C3aR1 were higher in carotid plaques compared to control arteries. Priming human carotid plaques with C5a followed by CC incubation resulted in pronounced release of IL-1ß, IL-18 and IL-1α. Additionally, mRNA profiling demonstrated that C5a and TNF priming followed by CC incubation upregulated plaque expression of NLRP3 inflammasome components. INTERPRETATION: We demonstrate that CC are important local- and systemic complement activators, and we reveal that the interaction between CC and complement could exert its effect by activating the NLRP3 inflammasome, thus promoting the progression of atherosclerosis.

Therapeutic drug monitoring of infliximab compared to standard clinical treatment with infliximab: study protocol for a randomised, controlled, open, parallel-group, phase IV study (the NOR-DRUM study).

BACKGROUND: Infliximab (INX) and other tumour necrosis factor inhibitors (TNFi) have revolutionised the treatment of several immune mediated inflammatory diseases. Still, many patients do not respond sufficiently to therapy or lose efficacy over time. The large interindividual variation in serum drug concentrations on standard doses and the development of anti-drug antibodies are thought to be major reasons for treatment failures. Therapeutic drug monitoring (TDM), an individualised treatment strategy based on systematic assessments of serum drug concentrations, has been proposed as a clinical tool to optimise efficacy of INX treatment. TDM seems reasonable both from a clinical and an economical point of view, but the effectiveness of this treatment strategy has not yet been demonstrated in randomised clinical trials. The NORwegian DRUg Monitoring study (NOR-DRUM) aims to assess the effectiveness of TDM, both with regard to the achievement of remission in patients starting INX treatment (part A) as well as to maintain disease control in patients on INX treatment (part B). METHODS: The NOR-DRUM study is a randomised, open, controlled, parallel-group, comparative, multi-centre, national, superiority, phase IV study with two separate parts, NOR-DRUM A and NOR-DRUM B. Patients with rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, ulcerative colitis, Crohn's disease and psoriasis are included. In both study parts participants are randomised 1:1 to either TDM of infliximab (intervention group) or to standard treatment with infliximab without knowledge of drug levels or ADAb status (control group). NOR-DRUM A will include 400 patients starting INX therapy. The primary outcome is remission at 30 weeks. In NOR-DRUM B, 450 patients on maintenance treatment with INX will be included. The primary endpoint is occurrence of disease worsening during the 52-week study period. DISCUSSION: As the first trial to assess the effectiveness, safety and cost-effectiveness of TDM in patients receiving TNFi for a range of immune mediated inflammatory diseases, we hope that the NOR-DRUM study will contribute to the advancement of evidence based personalised treatment with biological medicines. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03074656. Registered on 090317.

Low Cellular NAD+ Compromises Lipopolysaccharide-Induced Inflammatory Responses via Inhibiting TLR4 Signal Transduction in Human Monocytes.

NAD+ is an essential cofactor in reduction-oxidation metabolism with impact on metabolic and inflammatory diseases. However, data elucidating the effects of NAD+ on the proinflammatory features of human primary monocytes are scarce. In this study, we explored how NAD+ affects TLR4 and NOD-like receptor with a PYD-domain 3 (NLRP3) inflammasome activation, two key innate immune responses. Human primary monocytes were isolated from buffy coats obtained from healthy individuals. Intracellular NAD+ was manipulated by nicotinamide riboside and the NAMPT inhibitor FK866. Cells were primed with LPS with or without subsequent NLRP3 activation with ATP or cholesterol crystals to analyze the effects of NAD+ levels on TLR4-mediated NF-κB activation and NLRP3 activity, respectively. Cytokine release was quantified, and the downstream signal pathway of TLR4 was investigated with Western blot and proteomic analysis. The impact of sirtuin and PARP inhibition was also explored. Our main findings were: 1) elevated NAD+ enhanced IL-1ß release in LPS-primed human monocytes exposed to ATP in vitro, 2) both NLRP3-dependent and -independent inflammatory responses in LPS-exposed monocytes were inhibited by NAD+ depletion with FK866, 3) the inhibition was not caused by suppression of sirtuins or PARP1, and 4) phosphorylation of several proteins TLR4 signal pathway was inhibited by FK866-mediated NAD+ depletion, specifically TAK1, IKKß, IkBα, MEK 1/2, ERK 1/2, and p38. Hence, we suggest a novel mechanism in which NAD+ affects TLR4 signal transduction. Furthermore, our data challenge previous reports of the interaction between NAD+ and inflammation and question the use of nicotinamide riboside in the therapy of inflammatory disorders.

Mammalian Target of Rapamycin (mTOR) and the Proteasome Attenuates IL-1ß Expression in Primary Mouse Cardiac Fibroblasts.

Background: IL-1ß is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1ß is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1ß. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1ß secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1ß release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1ß release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1ß release from cardiac fibroblasts. Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1ß secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1ß protein levels as well as LPS/ATP-induced IL-1ß release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1ß protein levels. Key findings were reinvestigated in human monocyte-derived macrophages. Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1ß synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine.

Interleukin 27 is increased in carotid atherosclerosis and promotes NLRP3 inflammasome activation.

AIM: Interleukin-27 (IL-27) is involved in different inflammatory diseases; however, its role in atherosclerosis is unclear. In this study we investigated the expression of IL-27 and its receptor in patients with carotid atherosclerosis and if IL-27 could modulate the inflammatory effects of the NLRP3 inflammasome in vitro. METHODS: Plasma IL-27 was measured by enzyme immunoassay in patients with carotid stenosis (n = 140) and in healthy controls (n = 19). Expression of IL-27 and IL-27R was analyzed by quantitative PCR and immunohistochemistry in plaques from patients and in non-atherosclerotic vessels. THP-1 monocytes, primary monocytes and peripheral blood mononuclear cells (PBMCs) were used to study effects of IL-27 in vitro. RESULTS: Our main findings were: (i) Plasma levels of IL-27 were significantly elevated in patients with carotid atherosclerotic disease compared to healthy controls. (ii) Gene expression of IL-27 and IL-27R was significantly elevated in plaques compared to control vessels, and co-localized to macrophages. (iii) In vitro, IL-27 increased NLRP3 inflammasome activation in monocytes with enhanced release of IL-1 ß. CONCLUSIONS: We demonstrate increased levels of IL-27 and IL-27R in patients with carotid atherosclerosis. Our in vitro findings suggest an inflammatory role for IL-27, which can possibly be linked to atherosclerotic disease development.

Toll-Like Receptor 9 Promotes Survival in SERCA2a KO Heart Failure Mice.

Aim. Inflammation is important in heart failure (HF). The role of the immune receptor toll-like receptor 9 (TLR9) in HF is not understood and not investigated in diastolic HF. We investigated the role of TLR9 in a murine diastolic HF model caused by cardiomyocyte SERCA2a excision. Methods and Results. We crossed SERCA2a KO and TLR9 KO mice to generate four mouse lines. Tamoxifen-induced cardiomyocyte SERCA2a gene excision was carried out in mice, causing diastolic HF. After 7.6 weeks, cardiac functions and dimensions were analyzed by echocardiography and heart tissues were processed. HF mice depleted of TLR9 demonstrated reduced survival compared to SERC2a KO mice, with a median life expectancy of 58 days compared to 63 days. Both HF groups displayed increased left atrium size, lung weight, fetal gene expressions, monocyte/macrophage infiltration, and fibrosis. However, there were no significant differences between the groups. Conclusion. In mice with SERCA2a KO-induced diastolic HF, the absence of TLR9 reduced median life expectancy. The cause remains elusive, as all investigated HF parameters were unaltered. Still, these findings support a salutary role of TLR9 in some subsets of HF conditions and underline the importance for future studies on the mechanisms of TLR9 in diastolic HF.

Palmitate promotes inflammatory responses and cellular senescence in cardiac fibroblasts.

Palmitate triggers inflammatory responses in several cell types, but its effects on cardiac fibroblasts are at present unknown. The aims of the study were to (1) assess the potential of palmitate to promote inflammatory signaling in cardiac fibroblasts through TLR4 and the NLRP3 inflammasome and (2) characterize the cellular phenotype of cardiac fibroblasts exposed to palmitate. We examined whether palmitate induces inflammatory responses in cardiac fibroblasts from WT, NLRP3-/- and ASC-/-mice (C57BL/6 background). Exposure to palmitate caused production of TNF, IL-6 and CXCL2 via TLR4 activation. NLRP3 inflammasomes are activated in a two-step manner. Whereas palmitate did not prime the NLRP3 inflammasome, it induced activation in LPS-primed cardiac fibroblasts as indicated by IL-1ß, IL-18 production and NLRP3-ASC co-localization. Palmitate-induced NLRP3 inflammasome activation in LPS-primed cardiac fibroblasts was associated with reduced AMPK activity, mitochondrial reactive oxygen species production and mitochondrial dysfunction. The cardiac fibroblast phenotype caused by palmitate, in an LPS and NLRP3 independent manner, was characterized by decreased cellular proliferation, contractility, collagen and MMP-2 expression, as well as increased senescence-associated ß-galactosidase activity, and consistent with a state of cellular senescence. This study establishes that in vitro palmitate exposure of cardiac fibroblasts provides inflammatory responses via TLR4 and NLRP3 inflammasome activation. Palmitate also modulates cardiac fibroblast functionality, in a NLRP3 independent manner, resulting in a phenotype related to cellular senescence. These effects of palmitate could be of importance for myocardial dysfunction in obese and diabetic patients.

Low Circulating Levels of Mitochondrial and High Levels of Nuclear DNA Predict Mortality in Chronic Heart Failure.

BACKGROUND: Mitochondrial DNA (mtDNA) and possibly nuclear DNA (nDNA) are released as danger-associated molecular patterns during cardiac stress, and may activate several innate immune receptors. The purpose of this study was to investigate the regulation of these danger-associated molecular patterns during human heart failure (HF). METHODS AND RESULTS: Plasma levels of mtDNA and nDNA from HF patients (n = 84) were analyzed by reverse transcriptase-polymerase chain reaction and compared with controls (n = 72). Increased levels of mtDNA were found in New York Heart Association (NYHA) I-II and NYHA III-IV. There was evidence of increased nDNA in NYHA III-IV compared with controls and NYHA I-II. Kaplan-Meier analysis revealed higher mortality in patients with high nDNA levels, whereas high levels of mtDNA were associated with survival. CONCLUSIONS: Plasma levels of mtDNA and nDNA are elevated in human HF associated with increased and decreased mortality, respectively. This study may suggest a rationale for exploring interventions within inflammatory signaling pathways activated by nucleic acids as novel targets in treatment of HF.

Sustained Toll-Like Receptor 9 Activation Promotes Systemic and Cardiac Inflammation, and Aggravates Diastolic Heart Failure in SERCA2a KO Mice.

AIM: Cardiac inflammation is important in the pathogenesis of heart failure. However, the consequence of systemic inflammation on concomitant established heart failure, and in particular diastolic heart failure, is less explored. Here we investigated the impact of systemic inflammation, caused by sustained Toll-like receptor 9 activation, on established diastolic heart failure. METHODS AND RESULTS: Diastolic heart failure was established in 8-10 week old cardiomyocyte specific, inducible SERCA2a knock out (i.e., SERCA2a KO) C57Bl/6J mice. Four weeks after conditional KO, mice were randomized to receive Toll-like receptor 9 agonist (CpG B; 2µg/g body weight) or PBS every third day. After additional four weeks, echocardiography, phase contrast magnetic resonance imaging, histology, flow cytometry, and cardiac RNA analyses were performed. A subgroup was followed, registering morbidity and death. Non-heart failure control groups treated with CpG B or PBS served as controls. Our main findings were: (i) Toll-like receptor 9 activation (CpG B) reduced life expectancy in SERCA2a KO mice compared to PBS treated SERCA2a KO mice. (ii) Diastolic function was lower in SERCA2a KO mice with Toll-like receptor 9 activation. (iii) Toll-like receptor 9 stimulated SERCA2a KO mice also had increased cardiac and systemic inflammation. CONCLUSION: Sustained activation of Toll-like receptor 9 causes cardiac and systemic inflammation, and deterioration of SERCA2a depletion-mediated diastolic heart failure.

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/ß). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Double blockade of CD14 and complement C5 abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice.

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)-induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54-95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24-48 h). Combined treatment increased median survival to 96 h (range 24-240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24-48 h] and 48 h [24-96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.