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Epigenetic Modulation of Microglial Inflammatory Gene Loci in Helminth-Induced Immune Suppression: Implications for Immune Regulation in Neurocysticercosis.

Chauhan A, Quenum FZ, Abbas A, Bradley DS, Nechaev S, Singh BB, Sharma J, Mishra BB - ASN Neuro (2015)

Bottom Line: Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia.Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well.These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, The University of North Dakota, Grand Forks, ND, USA.

No MeSH data available.


Related in: MedlinePlus

HSF-mediated modulation in Pol-II recruitment. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-Pol-II or isotype IgG or with no antibody. Pol-II occupancy at the promoter regions of CIITA (A), MHC-II (H2Eβ) (B), NOS-2 (C), TNF-α (D), IL-6(E), and Arginase-1 (F) was assessed by qPCR and expressed as percentage of input. The mean ± SE of Pol-II occupancy using anti-Pol-II or isotype IgG (control) in three independent experiments was determined. Significant differences were measured by Student’s t test (*p < .05; **p < .005; p < .001). HSF = helminth soluble/secreted factor; Pol-II = polymerase II; LPS = lipopolysaccharide; ChIP = Chromatin Immunoprecipitation; MHC = major histocompatibility complex; NOS2 = nitric oxide synthase 2; TNF-α = tumor necrosis factor-alpha; IL-6 = interleukin-6; qPCR = quantitative polymerase chain reaction.
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fig3-1759091415592126: HSF-mediated modulation in Pol-II recruitment. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-Pol-II or isotype IgG or with no antibody. Pol-II occupancy at the promoter regions of CIITA (A), MHC-II (H2Eβ) (B), NOS-2 (C), TNF-α (D), IL-6(E), and Arginase-1 (F) was assessed by qPCR and expressed as percentage of input. The mean ± SE of Pol-II occupancy using anti-Pol-II or isotype IgG (control) in three independent experiments was determined. Significant differences were measured by Student’s t test (*p < .05; **p < .005; p < .001). HSF = helminth soluble/secreted factor; Pol-II = polymerase II; LPS = lipopolysaccharide; ChIP = Chromatin Immunoprecipitation; MHC = major histocompatibility complex; NOS2 = nitric oxide synthase 2; TNF-α = tumor necrosis factor-alpha; IL-6 = interleukin-6; qPCR = quantitative polymerase chain reaction.

Mentions: An important step in the regulation of gene expression is the recruitment of Pol-II along with the general transcription factors that enable the initiation of RNA synthesis (Rogatsky and Adelman, 2014). We next examined the effect of HSF on the recruitment of Pol-II to the promoter of MHC-II, CIITA, TNF-α, IL-6, and NOS2 using ChIP assays. As positive control, the level of Pol-II occupancy at the housekeeping GAPDH in the same sample was measured. As negative control, ChIP analysis using isotype IgG antibody was performed on the same samples. Compared with the untreated microglia, HSF-stimulated cells displayed a significantly decreased Pol-II occupancy at the promoter of all the immune mediators analyzed (MHC-II, CIITA, TNF-α, IL-6, and NOS2; Figure 3(A) to (E)). The effect of HSF on LPS-induced Pol-II recruitment to the promoter of the previously mentioned host mediators was examined. As expected, exposure to LPS significantly increased Pol-II occupancy at the promoter regions of MHC-II (Figure 3(B)), CIITA (Figure 3(A)), NOS2 (Figure 3(C)),TNF-α (Figure 3(D)), and IL-6 (Figure 3(E)), whereas minimal to no Pol-II occupancy was observed using the isotype IgG antibodies (data not shown). This effect of LPS was significantly inhibited in the presence of HSF (Figure 3). In contrast, HSF, LPS, HSF/LPS stimulation had no significant effect on Pol-II occupancy at the housekeeping gene GAPDH, as compared with the untreated microglia (data not shown). Moreover, as helminth infections are known to induce markers associated with M2 in helminth infections (Harn et al., 2009; Jenkins and Allen, 2010; Mishra et al., 2010), Pol-II occupancy was examined at the promoter regions of Arginase-1 (a signature marker of M2 activation phenotype). As compared with control cells, HSF-stimulated cells displayed a significantly increased Pol-II occupancy at the promoter of Arginase-1 (Figure 3(F)). In contrast, no difference in Pol-II occupancy was observed at the promoter of Arginase-1 after LPS exposure, which was significantly augmented in the presence of HSF (Figure 3(F)). Taken together, results from these experiments suggest that HSF inhibited Pol-II recruitment at the promoter of the inflammatory immune mediators in microglia.Figure 3.


Epigenetic Modulation of Microglial Inflammatory Gene Loci in Helminth-Induced Immune Suppression: Implications for Immune Regulation in Neurocysticercosis.

Chauhan A, Quenum FZ, Abbas A, Bradley DS, Nechaev S, Singh BB, Sharma J, Mishra BB - ASN Neuro (2015)

HSF-mediated modulation in Pol-II recruitment. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-Pol-II or isotype IgG or with no antibody. Pol-II occupancy at the promoter regions of CIITA (A), MHC-II (H2Eβ) (B), NOS-2 (C), TNF-α (D), IL-6(E), and Arginase-1 (F) was assessed by qPCR and expressed as percentage of input. The mean ± SE of Pol-II occupancy using anti-Pol-II or isotype IgG (control) in three independent experiments was determined. Significant differences were measured by Student’s t test (*p < .05; **p < .005; p < .001). HSF = helminth soluble/secreted factor; Pol-II = polymerase II; LPS = lipopolysaccharide; ChIP = Chromatin Immunoprecipitation; MHC = major histocompatibility complex; NOS2 = nitric oxide synthase 2; TNF-α = tumor necrosis factor-alpha; IL-6 = interleukin-6; qPCR = quantitative polymerase chain reaction.
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fig3-1759091415592126: HSF-mediated modulation in Pol-II recruitment. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-Pol-II or isotype IgG or with no antibody. Pol-II occupancy at the promoter regions of CIITA (A), MHC-II (H2Eβ) (B), NOS-2 (C), TNF-α (D), IL-6(E), and Arginase-1 (F) was assessed by qPCR and expressed as percentage of input. The mean ± SE of Pol-II occupancy using anti-Pol-II or isotype IgG (control) in three independent experiments was determined. Significant differences were measured by Student’s t test (*p < .05; **p < .005; p < .001). HSF = helminth soluble/secreted factor; Pol-II = polymerase II; LPS = lipopolysaccharide; ChIP = Chromatin Immunoprecipitation; MHC = major histocompatibility complex; NOS2 = nitric oxide synthase 2; TNF-α = tumor necrosis factor-alpha; IL-6 = interleukin-6; qPCR = quantitative polymerase chain reaction.
Mentions: An important step in the regulation of gene expression is the recruitment of Pol-II along with the general transcription factors that enable the initiation of RNA synthesis (Rogatsky and Adelman, 2014). We next examined the effect of HSF on the recruitment of Pol-II to the promoter of MHC-II, CIITA, TNF-α, IL-6, and NOS2 using ChIP assays. As positive control, the level of Pol-II occupancy at the housekeeping GAPDH in the same sample was measured. As negative control, ChIP analysis using isotype IgG antibody was performed on the same samples. Compared with the untreated microglia, HSF-stimulated cells displayed a significantly decreased Pol-II occupancy at the promoter of all the immune mediators analyzed (MHC-II, CIITA, TNF-α, IL-6, and NOS2; Figure 3(A) to (E)). The effect of HSF on LPS-induced Pol-II recruitment to the promoter of the previously mentioned host mediators was examined. As expected, exposure to LPS significantly increased Pol-II occupancy at the promoter regions of MHC-II (Figure 3(B)), CIITA (Figure 3(A)), NOS2 (Figure 3(C)),TNF-α (Figure 3(D)), and IL-6 (Figure 3(E)), whereas minimal to no Pol-II occupancy was observed using the isotype IgG antibodies (data not shown). This effect of LPS was significantly inhibited in the presence of HSF (Figure 3). In contrast, HSF, LPS, HSF/LPS stimulation had no significant effect on Pol-II occupancy at the housekeeping gene GAPDH, as compared with the untreated microglia (data not shown). Moreover, as helminth infections are known to induce markers associated with M2 in helminth infections (Harn et al., 2009; Jenkins and Allen, 2010; Mishra et al., 2010), Pol-II occupancy was examined at the promoter regions of Arginase-1 (a signature marker of M2 activation phenotype). As compared with control cells, HSF-stimulated cells displayed a significantly increased Pol-II occupancy at the promoter of Arginase-1 (Figure 3(F)). In contrast, no difference in Pol-II occupancy was observed at the promoter of Arginase-1 after LPS exposure, which was significantly augmented in the presence of HSF (Figure 3(F)). Taken together, results from these experiments suggest that HSF inhibited Pol-II recruitment at the promoter of the inflammatory immune mediators in microglia.Figure 3.

Bottom Line: Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia.Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well.These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Basic Sciences, School of Medicine and Health Sciences, The University of North Dakota, Grand Forks, ND, USA.

No MeSH data available.


Related in: MedlinePlus