Specific Inflammatory Stimuli Lead to Distinct Platelet Responses in Mice and Humans

INTRODUCTION: Diverse and multi-factorial processes contribute to the progression of cardiovascular disease. These processes affect cells involved in the development of this disease in varying ways, ultimately leading to atherothrombosis. The goal of our study was to compare the differential effects of specific stimuli - two bacterial infections and a Western diet - on platelet responses in ApoE-/- mice, specifically examining inflammatory function and gene expression. Results from murine studies were verified using platelets from participants of the Framingham Heart Study (FHS; n = 1819 participants). METHODS: Blood and spleen samples were collected at weeks 1 and 9 from ApoE-/- mice infected with Porphyromonas gingivalis or Chlamydia pneumoniae and from mice fed a Western diet for 9 weeks. Transcripts based on data from a Western diet in ApoE-/- mice were measured in platelet samples from FHS using high throughput qRT-PCR. RESULTS:At week 1, both bacterial infections increased circulating platelet-neutrophil aggregates. At week 9, these cells individually localized to the spleen, while Western diet resulted in increased platelet-neutrophil aggregates in the spleen only. Microarray analysis of platelet RNA from infected or Western diet-fed mice at week 1 and 9 showed differential profiles. Genes, such as Serpina1a, Ttr, Fgg, Rpl21, and Alb, were uniquely affected by infection and diet. Results were reinforced in platelets obtained from participants of the FHS. CONCLUSION: Using both human studies and animal models, results demonstrate that variable sources of inflammatory stimuli have the ability to influence the platelet phenotype in distinct ways, indicative of the diverse function of platelets in thrombosis, hemostasis, and immunity

Distinct gene signatures in aortic tissue from ApoE-/- mice exposed to pathogens or Western diet

BACKGROUND: Atherosclerosis is a progressive disease characterized by inflammation and accumulation of lipids in vascular tissue. Porphyromonas gingivalis (Pg) and Chlamydia pneumoniae (Cp) are associated with inflammatory atherosclerosis in humans. Similar to endogenous mediators arising from excessive dietary lipids, these Gram-negative pathogens are pro-atherogenic in animal models, although the specific inflammatory/atherogenic pathways induced by these stimuli are not well defined. In this study, we identified gene expression profiles that characterize P. gingivalis, C. pneumoniae, and Western diet (WD) at acute and chronic time points in aortas of Apolipoprotein E (ApoE-/-) mice. RESULTS: At the chronic time point, we observed that P. gingivalis was associated with a high number of unique differentially expressed genes compared to C. pneumoniae or WD. For the top 500 differentially expressed genes unique to each group, we observed a high percentage (76%) that exhibited decreased expression in P. gingivalis-treated mice in contrast to a high percentage (96%) that exhibited increased expression in WD mice. C. pneumoniae treatment resulted in approximately equal numbers of genes that exhibited increased and decreased expression. Gene Set Enrichment Analysis (GSEA) revealed distinct stimuli-associated phenotypes, including decreased expression of mitochondrion, glucose metabolism, and PPAR pathways in response to P. gingivalis but increased expression of mitochondrion, lipid metabolism, carbohydrate and amino acid metabolism, and PPAR pathways in response to C. pneumoniae; WD was associated with increased expression of immune and inflammatory pathways. DAVID analysis of gene clusters identified by two-way ANOVA at acute and chronic time points revealed a set of core genes that exhibited altered expression during the natural progression of atherosclerosis in ApoE-/- mice; these changes were enhanced in P. gingivalis-treated mice but attenuated in C. pneumoniae-treated mice. Notable differences in the expression of genes associated with unstable plaques were also observed among the three pro-atherogenic stimuli. CONCLUSIONS: Despite the common outcome of P. gingivalis, C. pneumoniae, and WD on the induction of vascular inflammation and atherosclerosis, distinct gene signatures and pathways unique to each pro-atherogenic stimulus were identified. Our results suggest that pathogen exposure results in dysregulated cellular responses that may impact plaque progression and regression pathways

Enhanced Virulence of Chlamydia muridarum Respiratory Infections in the Absence of TLR2 Activation

Chlamydia trachomatis is a common sexually transmitted pathogen and is associated with infant pneumonia. Data from the female mouse model of genital tract chlamydia infection suggests a requirement for TLR2-dependent signaling in the induction of inflammation and oviduct pathology. We hypothesized that the role of TLR2 in moderating mucosal inflammation is site specific. In order to investigate this, we infected mice via the intranasal route with C. muridarum and observed that in the absence of TLR2 activation, mice had more severe disease, higher lung cytokine levels, and an exaggerated influx of neutrophils and T-cells into the lungs. This could not be explained by impaired bacterial clearance as TLR2-deficient mice cleared the infection similar to controls. These data suggest that TLR2 has an anti-inflammatory function in the lung during Chlamydia infection, and that the role of TLR2 in mucosal inflammation varies at different mucosal surfaces

Assessment of COVID-19 vaccine take-up and its predictors among healthcare professionals in public hospitals, Addis Ababa, Ethiopia: Facility-based cross-sectional study

Healthcare professionals (HCPs) are among the highly exposed groups for the COVID-19 pandemic and have been identified as the target population to get vaccination against the spread of the infection. Aimed to assess COVID-19 vaccine take-up and its predictors among HCPs in public hospitals in Addis Ababa, Ethiopia, 2021. A facility-based cross-sectional study was conducted among 403 randomly selected participants from October 1st to November 30, 2021. Data was entered into Epi-info version 7 and analyzed using SPSS version 25. An AOR along with a 95% confidence level was estimated, and a P value <.05 was considered to declare the statistical significance. About 71% of the participants had taken any of the COVID-19 vaccines at least once. Being married (AOR: 10.79; 95% CI: 1.32–18.05); educational status of MSc degree (AOR = 7.7; CI: 2.08–15.1.6), medical doctors/GP (AOR = 5.88; CI: 1.60–15.54), MD with MSc (AOR = 9.63; CI: 2.17–17.76), PHD (AOR = 13.33; CI: 1.23–24.21) and specialist and above 3 (AOR = 3.45; CI: 2.34–42.8) holder HCPs; perceived severity of COVID-19 infection as moderate (AOR = 0.23; CI: 0.08–0.65) and mild (AOR = 0.28; CI: 0.11–0.74) and poor knowledge toward COVID-19 vaccination (AOR = 0.03; CI: 0.01–0.12) were statistically associated. This study showed that COVID-19 vaccine take-up among HCPs was relatively low to achieve herd immunity. Participants’ marital status, educational status, perception of the severity of COVID-19 infection, and level of knowledge toward COVID-19 vaccines were the predictors of the COVID-19 vaccine take-up

Intranasal infection with plasmid-cured strains of <i>C. muridarum</i> induces more exaggerated lung inflammatory cytokine response compared to wild type <i>C. muridarum</i> infected mice.

<p>Mice were inoculated via the intranasal route with <i>C. muridarum</i> Nigg or one of the plasmid-deficient mutant strains, CM972 or CM3.1, or mock infected with PBS, as described in the Methods. At seven days post infection, lung homogenates were assayed for a panel of 22 inflammatory cytokines and chemokines. Shown above are representative results for 6 of the cytokines assayed. Each data point represents one mouse, and the horizontal bar represents the mean. Significance is as follows: *, p≤0.05; **, p≤0.01; ***, p≤0.001; and ****, p≤0.001 for the wild type Nigg vs. plasmid mutant 972 or 3.1 infected mice using an unpaired <i>t</i>-test. This figure is representative of two independent experiments performed.</p

TLR2 deficiency leads to exaggerated PMN and T cell response in the lungs of C. muridarum infected mice.

<p>C57BL/6 or TLR2 KO mice on the same background were intranasally inoculated with 5×10³ IFU/mouse of <i>C. muridarum</i> Nigg. At the indicated day post infection, lungs from infected mice were processed to evaluate inflammatory cell infiltrates. (A) At day 5 and day 7 post-infection, lungs were fixed, sectioned and stained using CAE to quantify the PMN infiltration, as described in the Methods. Significance was calculated using an unpaired <i>t</i>-test, and the p-value is noted. (B) Single-cell suspensions of infected lungs at day 7 were prepared, stained, and analyzed by flow cytometry, as described in the Methods. F4/80-PE Cy5 was used as a marker for macrophages; Gr-1-PE Cy5 for neutrophils; CD19-PE for B lymphocytes and CD3-PE for T lymphocytes. The bar graph represents the percentage of positive cells from total lung cells, as determined by histogram data. Significance is as follows: *, p≤0.05; **, p≤0.01 for the infected C57BL/6 vs. infected TLR2-deficient mice using an unpaired <i>t</i>-test. This figure is representative of two independent experiments performed.</p

Intranasal infection with <i>C. muridarum</i> induces more extensive inflammatory changes in the lungs of TLR2-deficient mice compared to wild type mice.

<p>C57BL/6 or TLR2 KO mice on the same background were intranasally inoculated with PBS (mock) or 5×10³ IFU/mouse of <i>C. muridarum</i> Nigg. At the indicated time points, mice were euthanized and lungs were removed for tissue processing, as described in the Methods. A–D and G show routine H&E staining; P = dense PMN infiltrate, shown as deep purple-staining cells. E–F and H show vimentin and CD19 staining by immunohistochemistry, respectively. (A) C57BL/6, day 7; (B) TLR2-deficient, day 7; (C) C57BL/6, day 14; (D), TLR2-deficient, day 14. (E–F) Lungs from infected TLR2-deficient mice (F) fail to stain for vimentin (V) at 5 days post infection compared to C57BL/6 mice (E). (G–H) Lungs from infected TLR2-deficient mice show evidence of iBALT (*) at 7 days post infection when stained with H&E (G) or anti CD19 Ab to detect CD19-expressing B cells (H). Original magnification 40×. This figure is representative of three independent experiments performed.</p

Intranasal infection of TLR2-deficient mice with <i>C. muridarum</i> induces more exaggerated lung inflammatory cytokine response compared to infected wild type mice.

<p>C57BL/6 or TLR2 KO mice on the same background were intranasally inoculated with PBS (mock) or 5×10³ IFU/mouse of <i>C. muridarum</i> Nigg, as described in the Methods. At seven days post infection, lung homogenates were assayed for a panel of 22 inflammatory cytokines and chemokines. Shown above are representative results for 6 of the cytokines assayed. Each data point represents one mouse, and the horizontal bar represents the mean. Significance was calculated as follows using a two-tailed <i>t</i>-test: **, p≤0.01 and ***, p≤0.001 for the infected C57BL/6 vs. infected TLR2-deficient mice. NS, not significant. This figure is representative of three independent experiments performed.</p

Cytokine induction in wild type vs. TLR2-deficient macrophages.

<p>(A) BMDM from C57BL/6 or TLR2 KO mice on the same background were left untreated, or infected with the indicated MOI of <i>C. muridarum</i> Nigg (Cm). Synthetic lipopeptide Pam₃Cys-Ser-Lys₄ (Pam3; 100 ng/ml) or LPS was used as a control. The supernatants were harvested at 24 hr post treatment and assayed for TNF-α and IP-10 by ELISA. The values showed are the mean ± SEM from triplicate samples. (B) BMDM and alveolar macrophages (AM) from C57BL/6 or TLR2 KO mice on the same background were infected with <i>C. muridarum</i> Nigg at MOI of 3∶1. LPS (100 ng/ml) was used as control. The supernatants were harvested at the indicated time points and assayed for TNF-α by ELISA. Significance was calculated as follows using a two-tailed <i>t</i>-test: *, p≤0.05; **, p≤0.01; ***, p≤0.001; ****, p≤0.0001.</p

Weight change and bacterial clearance of <i>C. muridarum</i> infected wild type vs. TLR2-deficient mice.

<p>C57BL/6 or TLR2 KO mice on the same background were intranasally inoculated with PBS (mock; N = 6) or 2.5×10⁴ IFU/mouse of <i>C. muridarum</i> Nigg (N = 8), as described in the Methods. (A) Mice were weighed daily for 9 days post infection. Shown above is the weight change relative to the starting weight of each individual mouse (weight day X/weight day 0). Significance is as follows: p = 0.0474 infected BL/6 vs infected TLR2 KO over time, using RM ANOVA as described in the methods using a two-factor RM ANOVA with post hoc test. No significant difference in weight was observed between uninfected strains. (B) Mice were infected with 5×10³ IFU <i>C. muridarum</i> Nigg, and were euthanized on day 2, 4, 7, 14 and day 20. Quantitative bacterial cultures were determined from whole lung homogenates, as described in the Methods. Quantitative culture data was confirmed by quantitative PCR (data not shown). Statistical analysis of the infectious burden over time using a Mann-Whitney U test was not significant. This figure is representative of three independent experiments performed.</p