PMC

E4 microglia exhibit increased interferon signaling, increased eIF2 signaling, and decreased protein translation at baseline. a) Expression of several genes from the interferon α/β and interferon ɣ signaling pathways () were measured from cell lysates of E3 and E4 microglia using qRT-PCR. E4 microglia exhibited an increase in the expression of several of these genes compared to E3 microglia; unpaired Welch’s t-test followed by Benjamini-Hochberg (BH) correction for multiple comparisons, Ifnαr1 avg. fold change (FC) ± SEM = 1.1 ± 0.4, adj. p = 1.0; Ifnαr2 FC = 3.0 ± 0.6, adj. p = 0.02; Irf9 FC = 1.1 ± 0.2, adj.p = 1.0; Irf2 FC = 0.31 ± 0.04, adj. p = 0.005; Irf5 FC = 1.4 ± 0.1, adj. p = 0.08, Irf8 FC = 4.8 ± 0.6, adj. p = 0.002; Stat1 FC = 2.8 ± 0.40, adj. p = 0.06; Stat2 avg. FC = 7.8 ± 0.85, adj. p = 0.0003; Oas1g FC = 1.6 ± 0.2, adj.p = 0.06; Oas2 avg. FC = 5.1 ± 0.60, adj. p = 0.0004; Oas3 avg. FC = 3.8 ± 0.29, adj p = 0.0004; Irf3 FC = 1.0 ± 0.08, adj. p = 1.0; Irf7 avg. FC = 11.7 ± 1.1, adj. p = 5.7 × 10⁻⁰⁵; Il1β FC = 1.0 ± 0.1, adj. p = 1.0; Tnfα avg. FC = 1.90 ± 0.07, adj. p = 0.02; Cxcl1 FC = 1.1 ± 0.2, adj. p = 1.0; Ccl2 avg. FC = 4.5 ± 0.41, adj. p = 0.0003; Il6 avg. FC = 8.8 ± 1.4, adj. p = 0.003, Ccl5 FC = 7.9 ± 4.0, adj. p = 0.06; Cxcl10 avg. FC = 213.5 ± 12.3, adj. p = 3.1 × 10⁻⁰⁷; Ikke avg. FC = 1.9 ± 0.1, adj. p = 0.01; Cxcl9: 669.5 ± 187.9, adj. p = 0.004; Jak1 FC = 0.7 ± 0.1, adj. p = 0.3; Irf1 avg. FC = 50.3 ± 3.1, adj. p = 6.05 × 10⁻⁰⁵. b) Levels of select secreted cytokines/chemokines and other inflammatory mediators were measured in the conditioned media from E3 and E4 microglial cultures using a Cytokine Proteome Profiler array kit. E4 microglia showed significantly increased levels of specific cytokines/chemokines compared to E3: unpaired Student’s t-test followed by BH correction for multiple comparisons, IL1α t(8) = 4.7, adj. p = 0.01, d = 3.0 CI_95% [1.0, 4.8]; CCL2 t(8) = 4.2, adj. p = 0.02, d = 2.7 CI_95% [0.8, 4.4]; CCL12 t(8) = 4.1, adj. p = 0.02, d = 2.6 CI_95% [0.8, 4.3]; CCL3 t(8) = 5.8, adj. p = 0.007, d = 3.6 CI_95% [1.5, 5.7]; CCL4 t(8) = 5.3, adj. p = 0.007, d = 3.4 CI_95% [1.3, 5.4]; CXCL2 t(8) = 5.3, adj. p = 0.007, d = 3.4, CI_95% [1.3, 5.4]; CCL17 t(8) = 1.5, adj. p = 0.8, d = 1.0 CI_95% [−0.4, 2.3]; TNFα t(8) = 5.3, adj. p = 0.007, d = 3.3 CI_95% [1.2, 5.3], n = 5 independent experiments. c) Levels of secreted interferons in the supernatant of E3 and E4 microglia cultures were measured using ELISA. E4 microglia showed no significant change in interferon-α/β; t(3.8) = −0.6, p = 0.6, d = −0.5 CI_95% [−1.8, 1.0], but exhibited a significant increase in interferon-ɣ t(2) = 10.8, p = 0.008, d = 8.8 CI_95% [2.8, 14.9] relative to E3 microglia. d) Protein synthesis was measured in E3 and E4 microglia using the SUnSET assay, which detects levels of puromycin incorporated into newly synthesized proteins. A representative western blot is shown. M indicates the molecular weight ladder and both short exposure (top) and longer exposure (bottom) are displayed. e) Original non-over exposed western blot (top) was quantified and shows decreased puromycin incorporation in E4 microglia at 1 hour compared to E3, t(3.0) = −8.6, p = 0.003, d = −6.1 CI_95% [−10.9, −1.4]. f) Representative Western blots showing the protein levels of both total and phosphorylated eIF2α, PRK, PERK, and GCN2 protein. g) Quantification of western blots shows E4 microglia exhibit a significant increase in the ratio of phosphorylated to total eIF2, t(4.8) = 4.6, p = 0.007, d = 3.2 CI_95% [0.8, 5.6] and PKR; t(3.0) = 8.6, p = 0.04, d = 2.5 CI_95% [0.1, 4.9] compared to E3 microglia. The ratio of p-PERK to total PERK was increased but did not reach statistical significance, t(3.0) = 2.7, p = 0.07, d = 1.9 CI_95% [−0.2, 3.9] while the percentage of GCN2 that was phosphorylated remained unchanged, t(3.3) = 0.1, p = 0.92, d = 0.1 CI_95% [−1.3, 1.5]. a, c, e, g) Each dot corresponds to an independent experiment (n = 4/group). Black horizontal bars indicate the mean and error bars represent SE. c, e,g) Unpaired Welch’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Relationship between the percentages of CO₂ refixed (P_r) and (a) mesophyll resistance to CO₂ diffusion (r_m), (b) stomatal resistance to CO₂ diffusion (r_s) and (c) LMA in six different woody species. Deciduous species are marked with circles, and evergreens are marked with triangles. Regression lines: (a) all species (black solid line, y = 36.7 + 5.8x, P < 0.01, adj. r² = 0.22), B. pendula (dashed red line, y = 38.7 + 4.0x, P = 0.22, adj. r² = 0.02), Q. robur (dashed gray line, y = 33.0 + 1.6x, P = 0.25, adj. r² = 0.02), L. decidua (dashed black line, y = 43.4 + −0.8x, P = 0.75, adj. r² = −0.04), P. sylvestris (dashed purple line, y = 40.6 ± 0.9x, P = 0.71, adj. r² = −0.06), P. abies (dashed yellow line, y = 47.3 + 5.5x, P = 0.16, adj. r² = 0.05) and V. vitis-idaea (dashed orange line, y = 37.3 + 7.5x, P < 0.01, adj. r² = 0.43); (b) all species (black solid line, y = 39.8 + 0.56x, P < 0.01, adj. r² = 0.17), B. pendula (dashed red line, y = 26.5 + 1.7x, P < 0.01, adj. r² = 0.784), Q. robur (dashed gray line, y = 31.5 + 0.38x, P = 0.011, adj. r² = 0.19), L. decidua (dashed black line, y = 37.6 + 0.62x, P = 0.14, adj. r² = 0.05), P. sylvestris (dashed purple line, y = 32.6 + 1.5x, P = 0.06, adj. r² = 0.12), P. abies (dashed yellow line, y = 51.7 + 0.4x, P = 0.01, adj. r² = 0.23) and V. vitis-idaea (dashed orange line, y = 50.9+ 0.51x, P = 0.5, adj. r² = −0.02) and (c) all species (black solid line, y = 41.6 + 0.034x, P = 0.012, adj. r² = 0.035), B. pendula (dashed red line, y = 30.7 + 0.23x, P = 0.13, adj. r² = 0.06), Q. robur (dashed gray line, y = 22.28 + 0.29x, P < 0.01, adj. r² = 0.40), L. decidua (dashed black line, y = 19.51 + 0.25x, P = 0.025, adj. r² = 0.15), P. sylvestris (dashed purple line, y = 44.6 – 0.01x, P = 0.76, adj. r² = −0.04), P. abies (dashed yellow line, y = 62.9 – 0.02x, P = 0.793, adj. r² = −0.04), V. vitis-idaea (dashed orange line, y = 67.5 – 0.11x, P = 0.344, adj. r² = −0.003). N = 136.

Results of the clinical colitis score. Clinical colitis score of all three groups of the DSS-induced colitis model (n = 10 animals in each group). Comparison to d0: one way RM ANOVA + Dunnett’s post hoc test;°: 0% [F₍₁₄, ₁₂₆₎ = 1.678, p = 0.0682; d8: adj. p = 0.0154], ∼-∼∼∼: 1.5% [F₍₁₄, ₁₂₆₎ = 4.094, p < 0.0001; d3: adj. p = 0.0428, d4: adj. p = 0.0004, d5: p = 0.0428, d6: adj. p = 0.0104], *-***: 2.5% [F₍₁₄, ₁₂₆₎ = 14.08, p < 0.0001; d2: adj. p = 0.0189, d3-6: adj. p < 0.0001, d7: adj. p = 0.0002, d8: adj. p = 0.0045, d9: p = 0.0189]. Comparison between the different groups: Kruskal-Wallis Test + Dunn’s post hoc test; #: 0% vs. 1.5% (d3: adj. p = 0.0237, d4: adj. p = 0.0101 and d6: adj. p = 0.0259), $-$$$: 0% vs. 2.5% (d2: p = 0.0015, d3-d6: p < 0.0001, d7: adj. p = 0.0008, d9: p = 0.0033, d10: p = 0.0154, d11: adj. p = 0.018), !, !!: 1.5% vs. 2.5% (d2: p = 0.0086, d5: p = 0.01).

Odds ratio graphs for severe respiratory failure (non-adjusted and adjusted). Legend: dotted line (------): non-adjusted logistic regression analyses. Continuous line ( ): adjusted logistic regression analyses for all significant variables. Variables evaluated without adjustment: Age > 80 = individuals aged 80 years or more; CKD = chronic kidney disease; CT scan = thomographic lung involvement over 50%; Marital status; Atrial Fib = prior atrial fibrillation, Vitamin D = vitamin D < 40 ng/dl; IL-6 = interleukin 6 > 40 pg/mL, CRP = C-reactive protein> 80 mg/L; D dimer = d dimer> 1000 ng/ml, Hemoglobin = hemoglobin< 12 g/mL; Leucocytes = leucocytes > 10.000 μL; Lymphocytes = lymphocytes < 600/μL; AST = aspartate aminotransferase > 50 U/mL; Creatinine = creatinine > 1.1 mg/dL; Myoglobin = myoglobin> 65 ng/mL). Variables evaluated with adjustment: Age > 80 Adj = individuals aged 80 years or more; CKD Adj = chronic kidney disease; CT scan Adj = lung involvement over 50%; Marital status Adj; Atrial Fib Adj = prior atrial fibrillation, Vitamin D Adj = vitamin D < 40 ng/dl; IL-6 Adj = interleukin 6 > 40 pg/mL, CRP Adj = C-reactive protein> 80 mg/L; D dimer Adj = d dimer> 1000 ng/ml, Hemoglobin Adj = hemoglobin< 12 g/mL; AST Adj = aspartate aminotransferase > 50 U/mL; Creatinine Adj = creatinine > 1.1 mg/dL; Myoglobin Adj = myoglobin> 65 ng/mL)