20 research outputs found
Beta interferon production is regulated by P38 mitogen-activated protein kinase in macrophages via both MSK1/2-and tristetraprolin-dependent pathways
Autocrine or paracrine signaling by beta interferon (IFN-β) is essential for many of the responses of macrophages to pathogen-associated molecular patterns. This feedback loop contributes to pathological responses to infectious agents and is therefore tightly regulated. We demonstrate here that macrophage expression of IFN-β is negatively regulated by mitogen- and stress-activated kinases 1 and 2 (MSK1/2). Lipopolysaccharide (LPS)-induced expression of IFN-β was elevated in both MSK1/2 knockout mice and macrophages. Although MSK1 and -2 promote the expression of the anti-inflammatory cytokine interleukin 10, it did not strongly contribute to the ability of MSKs to regulate IFN-β expression. Instead, MSK1 and -2 inhibit IFN-β expression via the induction of dual-specificity phosphatase 1 (DUSP1), which dephosphorylates and inactivates the mitogen-activated protein kinases p38 and Jun N-terminal protein kinase (JNK). Prolonged LPS-induced activation of p38 and JNK, phosphorylation of downstream transcription factors, and overexpression of IFN-β mRNA and protein were similar in MSK1/2 and DUSP1 knockout macrophages. Two distinct mechanisms were implicated in the overexpression of IFN-β: first, JNKmediated activation of c-jun, which binds to the IFN-β promoter, and second, p38-mediated inactivation of the mRNA-destabilizing factor tristetraprolin, which we show is able to target the IFN-β mRNA
SARS-CoV-2 Vaccine Responses in Individuals with Antibody Deficiency: Findings from the COV-AD Study
BACKGROUND: Vaccination prevents severe morbidity and mortality from COVID-19 in the general population. The immunogenicity and efficacy of SARS-CoV-2 vaccines in patients with antibody deficiency is poorly understood. OBJECTIVES: COVID-19 in patients with antibody deficiency (COV-AD) is a multi-site UK study that aims to determine the immune response to SARS-CoV-2 infection and vaccination in patients with primary or secondary antibody deficiency, a population that suffers from severe and recurrent infection and does not respond well to vaccination. METHODS: Individuals on immunoglobulin replacement therapy or with an IgG less than 4 g/L receiving antibiotic prophylaxis were recruited from April 2021. Serological and cellular responses were determined using ELISA, live-virus neutralisation and interferon gamma release assays. SARS-CoV-2 infection and clearance were determined by PCR from serial nasopharyngeal swabs. RESULTS: A total of 5.6% (n = 320) of the cohort reported prior SARS-CoV-2 infection, but only 0.3% remained PCR positive on study entry. Seropositivity, following two doses of SARS-CoV-2 vaccination, was 54.8% (n = 168) compared with 100% of healthy controls (n = 205). The magnitude of the antibody response and its neutralising capacity were both significantly reduced compared to controls. Participants vaccinated with the Pfizer/BioNTech vaccine were more likely to be seropositive (65.7% vs. 48.0%, p = 0.03) and have higher antibody levels compared with the AstraZeneca vaccine (IgGAM ratio 3.73 vs. 2.39, p = 0.0003). T cell responses post vaccination was demonstrable in 46.2% of participants and were associated with better antibody responses but there was no difference between the two vaccines. Eleven vaccine-breakthrough infections have occurred to date, 10 of them in recipients of the AstraZeneca vaccine. CONCLUSION: SARS-CoV-2 vaccines demonstrate reduced immunogenicity in patients with antibody deficiency with evidence of vaccine breakthrough infection
IFNβ autocrine feedback is required to sustain TLR induced production of MCP-1 in macrophages
AbstractChemokines, including MCP-1, are crucial to mounting an effective immune response due to their ability to recruit other immune cells. We show that sustained LPS or poly(I:C)-stimulated MCP-1 production requires an IFNβ-mediated feedback loop. Consistent with this, exogenous IFNβ was able to induce MCP-1 transcription in the absence of other stimuli. Blocking IFNβ signaling with Ruxolitinib, a JAK inhibitor, inhibited MCP-1 transcription. The MCP-1 promoter contains potential STAT binding sites and we demonstrate that STAT1 is recruited upon IFNβ stimulation. Furthermore we find that IL-10 knockout increases MCP-1 production in response to LPS, which may reflect an ability of IL-10 to repress IFNβ production. Overall, these results show the importance of the balance between IFNβ and IL-10 in the regulation of MCP-1
MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production via an interleukin-10 feedback loop
Prostaglandin production is catalyzed by cyclooxygenase 2 (cox-2). We demonstrate here that MSK1 and MSK2 (MSK1/2) can exert control on the induction of cox-2 mRNA by Toll-like receptor (TLR) agonists. In the initial phase of cox-2 induction, MSK1/2 knockout macrophages confirmed a role for MSK in the positive regulation of transcription. However, at later time points both lipopolysaccharide (LPS)-induced prostaglandin and cox-2 protein levels were increased in MSK1/2 knockout. Further analysis found that while MSKs promoted cox-2 mRNA transcription, following longer LPS stimulation MSKs also promoted degradation of cox-2 mRNA. This was found to be the result of an interleukin 10 (IL-10) feedback mechanism, with endogenously produced IL-10 promoting cox-2 degradation. The ability of IL-10 to do this was dependent on the mRNA binding protein TTP through a p38/MK2-mediated mechanism. As MSKs regulate IL-10 production in response to LPS, MSK1/2 knockout results in reduced IL-10 secretion and therefore reduced feedback from IL-10 on cox-2 mRNA stability. Following LPS stimulation, this increased mRNA stability correlated to an elevated induction of both of cox-2 protein and prostaglandin secretion in MSK1/2 knockout macrophages relative to that in wild-type cells. This was not restricted to isolated macrophages, as a similar effect of MSK1/2 knockout was seen on plasma prostaglandin E2 (PGE2) levels following intraperitoneal injection of LPS
SphK1 and LIGHT mRNA induction is regulated by Syk and IL-10.
<p>(A, B) Wild type BMDMs were stimulated with 200 µg/ml zymosan in the presence or absence of 4 µM Syk Inh II. The induction of SphK1 (A) and LIGHT (B) mRNA was determined by qPCR. (C, D) Wild type or IL-10 knockout BMDMs were stimulated for 8 h with 200 µg/ml zymosan and the induction of SphK1 (C) and LIGHT (D) mRNA was determined by qPCR. (E, F) Wild type or MSK1/2 knockout BMDMs were stimulated for 8 h with 200 µg/ml zymosan and the induction of SphK1 (E) and LIGHT (F) mRNA was determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **.</p
Zymosan induced MAPK activation and IL-10 production in part through dectin-1.
<p>(A) BMDMs were isolated from wild type or dectin-1 knockout animals on a C57/Bl6 background and stimulated with 200 µg/ml zymosan for 8 h. The levels of IL-10 protein secreted into the media were measured. (B) Wild type BMDMs were treated with 4 µM Syk Inhibitor II where indicated for 1 h before stimulation with 200 µg/ml zymosan for 8 h. Secreted IL-10 levels were then determined. In A and B error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less that 0.001 are indicated by **. (C) BMDMs from wild type or dectin-1 knockout on a 129SvJ background were stimulated for the indicated times with 200 µg/ml zymosan. The levels of phospho and total ERK1/2, phospho and total p38 and phospho CREB/ATF1 were then determined by immunoblotting. (D) Wild type BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 where indicated for 1 h before stimulation with 200 µg/ml zymosan for 30 min. The levels of total and phospho (T581) MSK1, phospho and total ERK1/2, phospho and total p38α and phospho CREB/ATF1 were then determined by immunoblotting. (E) Wild type or MSK1/2 double knockout BMDMs were stimulated with 200 µg/ml zymosan for the indicated times, and protein levels determined as in (D). In addition to MSK1, the total MSK1 antibody also picks up a non-specific band (ns) that runs at a molecular weight just below MSK1. The blots shown are representative of one (C) or three (D and E) separate experiments.</p
MSKs regulate IL-10 mRNA transcription via CREB.
<p>BMDMs were cultured from wild type, MSK1/2 knockout, CREB S133A knockin or MSK1/2 knockout/CREB S133A knockin mice. Cells were then stimulated with 200 µg/ml zymosan for either 1 h or for the indicated times. The induction of nur77 (A), TNFα (B) and IL-10 (C) mRNA was determined by qPCR. (D) Wild type or MSK1/2 knockout BMDMs were pretreated with 10 µM SB-747651A for 1 h where indicated and then stimulated with zymosan for 1 h. IL-10 mRNA induction was determined by qPCR. (E) MSK1/2 knockout BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 as indicated before stimulation with zymosan for 1 h. IL-10 mRNA induction was then determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. In A to C, a P value (Student's t-test) relative to the wild type cells of less than 0.05 is indicated by * and less than 0.01 by **. In D and E a P value of less than 0.01 relative to the no inhibitor control is indicated by **.</p
Dectin-1 specific ligands activate MSKs.
<p>(A) BMDMs from wild type or dectin-1 knockout on a 129SvJ background were treated with 10 µg/ml of curdlan for the indicated times and the levels of phospho and total ERK1/2, phospho and total p38α and phospho CREB/ATF1 determined by immunoblotting. (B) As (A) except cells were stimulated with 200 µg/ml of depleted zymosan. (C) Where indicated, wild type BMDMs were treated with 2 µM PD184352 or 5 µM SB203580 for 1 h. Cells were then stimulated with 10 µg/ml of curdlan for 30 min and the levels of total and phospho (T581) MSK1, phospho ERK1/2, phospho and total p38α and phospho CREB/ATF1 determined by immunoblotting. (D) Wild type or MSK1/2 knockout BMDMs were stimulated with 10 µg/ml of curdlan for the indicated times and the levels of the indicated proteins determined by immunoblotting. The blots shown are representative of one (B) or two (A, C, D) separate experiments.</p
MSKs promote IL-10 transcription.
<p>Wild type and MSK1/2 knockout BMDMs were isolated and stimulated with either 200 µg/ml of zymosan (A, B, C), 10 µg/ml curdlan (D, E, F) or 200 µg/ml of depleted zymosan (Dep Zym, G, H, I) for the indicated times. IL-10 protein levels secreted into the media were measured (A, D, G) and IL-10 mRNA (B, E, H) or IL-10 1° transcript (C, F, I) induction determined by qPCR. Error bars represent the standard deviation of independent cultures from 4 mice per genotype. P values (Student's t-test) of less than 0.05 are indicated by * and less than 0.01 by **. ns indicates a p value of more than 0.05.</p