Fig 2: Inhibition of TNF-a-induced cytokine gene activation by PLCe deficiency in primary-cultured bronchial epithelial cells.(A) Assessment of PLCe expression in primary-cultured bronchial epithelial cells was by RT-PCR. Primary cultures of bronchial epithelial cells were prepared from naïve adult PLCe +/+ mice. RNA was prepared for the first-strand preparation with (+) or without (-) reverse transcriptase (RTase) as indicated. (B) Effects of the PLCe genotype on Ccl2 and Cxcl2 expression induced by TNF-a. Primary cultures of bronchial epithelial cells prepared from naïve adult PLCe +/+ (filled bars) and PLCe ?X/?X (open bars) mice were treated without (-) or with (+) 10 ng/ml TNF-a for 3 h. RNA was purified and subjected to qRT-PCR to determine the mRNA levels of Ccl2 and Cxcl2. Data are representative of three independent experiments and expressed as the mean ± SD obtained by triplicate determinations. *, p<0.05; **, p<0.01; ***, p<0.001 between PLCe +/+ (filled bars) and PLCe ?X/?X (open bars) cells stimulated with TNF-a. (C) Effects of intracellular signaling inhibitors on Ccl2 expression induced by TNF-a stimulation. Primary-cultured PLCe +/+ bronchial epithelial cells prepared as in (B) were pretreated with dimethyl sulfoxide vehicle alone (-), 3 µM IKK inhibitor III (IKK), or 5 µM U73122 (U) for 10 min. Subsequently, they were stimulated with 10 ng/ml TNF-a for 3 h. QRT-PCR was carried out to determine the Ccl2 mRNA level, and data are presented as in (B). ***, p<0.001; n.s., statistically not significant.

Fig 3: Absence of CXCL2 and CXCL10 resulted in reduced intraocular inflammation during experimental Bacillus endophthalmitis. MPO concentrations were significantly reduced in the absence of CXCL2 and CXCL10. C57BL/6J (C57), CXCL2-/-, and CXCL10-/- mouse eyes were injected with 100 CFU B. cereus. At 8, 10, 12, 14, and 16 hours postinfection, infected eyes were harvested and infiltration of PMN was assessed by quantifying MPO in whole eyes by sandwich ELISA. At 8, 10, and 12 hours postinfection, MPO levels were significantly reduced (P = 0.05) in CXCL2-/- and CXCL10-/- eyes compared to that of C57 eyes. At 14 and 16 hours postinfection, MPO levels were significantly reduced in CXCL2-/- eyes compared to that of C57 eyes. No difference in MPO levels between CXCL10 and C57 were found at these time points. Compared to CXCL10-/-, CXCL2-/- eyes had reduced levels of MPO at 12 and 14 hours postinfection. Values represent means ± SEM for n = 5 eyes per group per time point with at least 3 independent experiments. *P = 0.05, **P = 0.01, and nsP = 0.05.

Fig 4: Absence of CXCL2 and CXCL10 did not affect intraocular Bacillus growth. Intraocular B. cereus ATCC 14579 growth was not affected by absence of CXCL2 or CXCL10. Eyes of CXCL2 -/-, CXCL10 -/-, and C57BL/6J (C57) male and female mice were infected with 100 CFU B. cereus. Eyes were harvested at designated timepoints and B. cereus were quantified. No significant differences in B. cereus burden were observed among mouse strains (P = 0.05) (A and C), and no difference was observed in intraocular bacterial burden between males and females (B and D). Values represent means ± SEM of n = 6 eyes at each time point with at least 3 independent experiments. nsP = 0.05.

Fig 5: Influence of TMZ on TAM infiltration, tumor vascularization, and expression of proangiogenic molecules in matched primary and recurrent GBMs. (a,b) Kaplan–Meier curves for (a) progression-free (PFS) and (b) overall survival (OS) of the subgroup analysis comparing the standard with the not standard group. (c–m) Graphs show the calculation of the stained area of TAMs (IBA1) (c–e), proangiogenic molecules VEGF (f), CXCL2 (g), IL8 (h) tumor vessels (CD31) (i–k), and CXCR2+ vessels (l,m) on FFPE tissue sections of primary and recurrent tumors of all patients. (c) Graph depicts the amount of TAMs in primary and recurrent tumors of both groups, which were significantly reduced in recurrent tumors (standard: mean difference: 246.7; 95% confidence interval -400.5 to -92.87; not standard: mean difference: -192.2; 95% confidence interval -370.0 to -14.40). (d,e) Graphs depict CXCL2+ TAMs in primary and recurrent tumors of both groups. (f–h) Graphs show calculation of the stained area of VEGF (f) and CXCL2 (g) (n = 38; standard: n = 18; not standard: n = 20). (h) The number of IL8+ patients in primary and recurrent tumors was calculated (n = 38; standard: n = 18; not standard: n = 20). (i–k) Graphs depict vessels/mm2 (i), the vessel area/vessel number (j), and vessel area/mm2 (k) (n = 30; standard: n = 15; not standard: n = 15). (l,m). The area of CXCR2+ vessels/CXCR2+ vessel number in (l) and the percentage of CXCR2+ vessels in relation to all vessels (m) was calculated (n = 28; standard: n = 13; not standard: n = 15). * p < 0.05, ** p < 0.01, *** p < 0.001. (c–m) paired Student’s t-test; box plots depicting mean (shown as “+”), median, 1st and 3rd quartile (shown as the box) and min to max (shown as the whiskers).