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The nicotinic receptor Alpha7 impacts the mouse lung response to LPS through multiple mechanisms.

Enioutina EY, Myers EJ, Tvrdik P, Hoidal JR, Rogers SW, Gahring LC - PLoS ONE (2015)

Bottom Line: This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n.LPS is significantly impaired.Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.

View Article: PubMed Central - PubMed

Affiliation: Geriatric Research, Education and Clinical Center (GRECC), Veterans Affairs Medical Center, Salt Lake City, Utah, United States of America; Department of Internal Medicine, Division of Geriatrics, University of Utah School of Medicine, Salt Lake City, Utah, United States of America; Department of Pathology, Division of Microbiology and Immunology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

ABSTRACT
The nicotinic acetylcholine receptor alpha7 (α7) is expressed by neuronal and non-neuronal cells throughout the body. We examined the mechanisms of the lung inflammatory response to intranasal (i.n.) lipopolysaccharide (LPS) regulated by α7. This was done in mice using homologous recombination to introduce a point mutation in the α7 receptor that replaces the glutamate residue 260 that lines the pore with alanine (α7E260A), which has been implicated in controlling the exceptional calcium ion conductance of this receptor. The α7E260A mice exhibit normal inflammatory cell recruitment to the blood in response to i.n. LPS administration. This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n. LPS is significantly impaired. While hematopoietic cells are recruited to the bloodstream in the α7E260A mouse, they fail to be recruited efficiently into both the interstitium and alveolar spaces of the lung. Bone marrow reconstitution experiments demonstrate that the responsiveness of both CD45+ and CD45- cells of the α7E260A mouse are impaired. The expression of several pro-inflammatory cytokine and chemokine RNAs including TNFα, IL-1α, Ccl2 and Cxcl10 are decreased in the α7E260A mouse. However, there is a substantial increase in IL-13 expression by CD45- lung interstitial cells in the α7E260A mouse. Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characteristics of BALF and interstitial cells from α7Cre:YFP mice challenged with i.n. LPS.Mice expressing cells that are lineage marked for WT α7 expression (α7Cre:YFP, 3–5 mo, 3–5 mice per group) were challenged with i.n. LPS (250 μg/mouse in 30 μl of saline). Control mice received i.n. 30 μl of saline. With LPS administration i.n., the number of cells isolated from the BALF and interstitium (CD45+) increases 2 to 3 fold by 72 hrs. A minimum of twenty thousand events were collected for each sample. Histograms reflect the percent of α7lin+ (YFP+) cells that are present on the days noted. The bar graphs represent data combined from three independent experiments (3–5 mice per experimental group in these experiments) with error bars reflecting plus-minus the standard error of the mean. Significance levels were determined using the Student’s t-test and this reflects the summed results of from at least three independent experiments. (A) BALF cells were isolated, FcRγ-blocked and stained with anti-mouse CD45, CD11c, and CD11b antibodies. In these experiments BALF was collected at Days 1, 3 and 8 for comparison to the naïve (saline control) group. Quantitative results are shown for day 1 and 3, where day 3 was observed to be the optimal response to LPS. Note the selective increase in the α7lin+ (YFP+) cell percentage after LPS exposure. (B, C) Cells of the interstitium were isolated from the remaining lung tissues by enzymatic digestion and stained with anti-mouse CD45, CD11b, EpCAM, and CD29. Flow cytometric gating was on either CD45+ cells (B) or on CD45- cells (C). The increase in percentage of CD45- α7lin+ (YFP+) cell after LPS exposure in this time frame was less in comparison to the CD45+ population. (D) Interstitial lung CD45+ and CD45- cells were collected 72 hrs post-challenge with either i.n. saline or LPS using CD45 microbeads and autoMacs sorting. RNA was isolated from these populations (CD45+ or CD45-). In both populations an increase in α7 RNA was observed in response to LPS administration. Combined analysis of α7 gene transcripts from three independent experiments is shown. Data are presented as mean ± SEM.
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pone.0121128.g002: Phenotypic characteristics of BALF and interstitial cells from α7Cre:YFP mice challenged with i.n. LPS.Mice expressing cells that are lineage marked for WT α7 expression (α7Cre:YFP, 3–5 mo, 3–5 mice per group) were challenged with i.n. LPS (250 μg/mouse in 30 μl of saline). Control mice received i.n. 30 μl of saline. With LPS administration i.n., the number of cells isolated from the BALF and interstitium (CD45+) increases 2 to 3 fold by 72 hrs. A minimum of twenty thousand events were collected for each sample. Histograms reflect the percent of α7lin+ (YFP+) cells that are present on the days noted. The bar graphs represent data combined from three independent experiments (3–5 mice per experimental group in these experiments) with error bars reflecting plus-minus the standard error of the mean. Significance levels were determined using the Student’s t-test and this reflects the summed results of from at least three independent experiments. (A) BALF cells were isolated, FcRγ-blocked and stained with anti-mouse CD45, CD11c, and CD11b antibodies. In these experiments BALF was collected at Days 1, 3 and 8 for comparison to the naïve (saline control) group. Quantitative results are shown for day 1 and 3, where day 3 was observed to be the optimal response to LPS. Note the selective increase in the α7lin+ (YFP+) cell percentage after LPS exposure. (B, C) Cells of the interstitium were isolated from the remaining lung tissues by enzymatic digestion and stained with anti-mouse CD45, CD11b, EpCAM, and CD29. Flow cytometric gating was on either CD45+ cells (B) or on CD45- cells (C). The increase in percentage of CD45- α7lin+ (YFP+) cell after LPS exposure in this time frame was less in comparison to the CD45+ population. (D) Interstitial lung CD45+ and CD45- cells were collected 72 hrs post-challenge with either i.n. saline or LPS using CD45 microbeads and autoMacs sorting. RNA was isolated from these populations (CD45+ or CD45-). In both populations an increase in α7 RNA was observed in response to LPS administration. Combined analysis of α7 gene transcripts from three independent experiments is shown. Data are presented as mean ± SEM.

Mentions: As observed in Fig. 1, naive α7lin+ and α7lin- cells exhibit similar marker expression (also see [28]). To determine if the α7lin+ or α7lin- differ in response to an inflammatory challenge, the response induced by intranasal administration of LPS was measured both in the blood and the different lung compartments (Fig. 2). Animal groups with similar initial α7lin+:α7lin- cell ratios as measured in the blood were administered either saline (i.n.) or LPS (i.n.) and the lungs harvested 24 hrs to 30 days later. We found that in three independent experiments the maximal response to LPS occurs at 72 hrs (Day 3) post LPS challenge (data not shown). Studies [18] using intratracheal administration of LPS report that the peak response in terms of cellular influx occurs at 48 hours which is likely due to the more direct, but more invasive, LPS challenge. We also found that the ratio of α7lin+:α7lin- cells present in BALF or interstitium of unstimulated animals (prior to any treatment) is less than measured in the blood. For example 18% of the cells in the blood are α7lin+ but in the BALF 12% are α7lin+ (Fig. 2A). However, the α7lin+/lin- ratio begins to increase to a statistically significant value relative to the saline control in both cells of the BALF and interstitium by 72 hours after LPS stimulation (Fig. 2A; p < 0.03). By day 8 the α7lin+:α7lin- ratio in both lung compartments returned to near pre-LPS levels where it remains to day 30 (Fig. 2A, B and data not shown). The lung samples from control (saline treated) mice were harvested at 72 hrs, and we observed no influx of inflammatory cells nor an increase in percentage of α7lin+ cells in the lungs at this time or at any of the saline treated points tested (Fig. 2 and not shown). YFP expression by the non-hematopoietic (CD45-) cells was also measured (Fig. 2C). There is a modest increase in the overall CD45- α7lin+:α7lin- ratio at 72 hours following LPS insult.


The nicotinic receptor Alpha7 impacts the mouse lung response to LPS through multiple mechanisms.

Enioutina EY, Myers EJ, Tvrdik P, Hoidal JR, Rogers SW, Gahring LC - PLoS ONE (2015)

Phenotypic characteristics of BALF and interstitial cells from α7Cre:YFP mice challenged with i.n. LPS.Mice expressing cells that are lineage marked for WT α7 expression (α7Cre:YFP, 3–5 mo, 3–5 mice per group) were challenged with i.n. LPS (250 μg/mouse in 30 μl of saline). Control mice received i.n. 30 μl of saline. With LPS administration i.n., the number of cells isolated from the BALF and interstitium (CD45+) increases 2 to 3 fold by 72 hrs. A minimum of twenty thousand events were collected for each sample. Histograms reflect the percent of α7lin+ (YFP+) cells that are present on the days noted. The bar graphs represent data combined from three independent experiments (3–5 mice per experimental group in these experiments) with error bars reflecting plus-minus the standard error of the mean. Significance levels were determined using the Student’s t-test and this reflects the summed results of from at least three independent experiments. (A) BALF cells were isolated, FcRγ-blocked and stained with anti-mouse CD45, CD11c, and CD11b antibodies. In these experiments BALF was collected at Days 1, 3 and 8 for comparison to the naïve (saline control) group. Quantitative results are shown for day 1 and 3, where day 3 was observed to be the optimal response to LPS. Note the selective increase in the α7lin+ (YFP+) cell percentage after LPS exposure. (B, C) Cells of the interstitium were isolated from the remaining lung tissues by enzymatic digestion and stained with anti-mouse CD45, CD11b, EpCAM, and CD29. Flow cytometric gating was on either CD45+ cells (B) or on CD45- cells (C). The increase in percentage of CD45- α7lin+ (YFP+) cell after LPS exposure in this time frame was less in comparison to the CD45+ population. (D) Interstitial lung CD45+ and CD45- cells were collected 72 hrs post-challenge with either i.n. saline or LPS using CD45 microbeads and autoMacs sorting. RNA was isolated from these populations (CD45+ or CD45-). In both populations an increase in α7 RNA was observed in response to LPS administration. Combined analysis of α7 gene transcripts from three independent experiments is shown. Data are presented as mean ± SEM.
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Related In: Results  -  Collection

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Show All Figures
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pone.0121128.g002: Phenotypic characteristics of BALF and interstitial cells from α7Cre:YFP mice challenged with i.n. LPS.Mice expressing cells that are lineage marked for WT α7 expression (α7Cre:YFP, 3–5 mo, 3–5 mice per group) were challenged with i.n. LPS (250 μg/mouse in 30 μl of saline). Control mice received i.n. 30 μl of saline. With LPS administration i.n., the number of cells isolated from the BALF and interstitium (CD45+) increases 2 to 3 fold by 72 hrs. A minimum of twenty thousand events were collected for each sample. Histograms reflect the percent of α7lin+ (YFP+) cells that are present on the days noted. The bar graphs represent data combined from three independent experiments (3–5 mice per experimental group in these experiments) with error bars reflecting plus-minus the standard error of the mean. Significance levels were determined using the Student’s t-test and this reflects the summed results of from at least three independent experiments. (A) BALF cells were isolated, FcRγ-blocked and stained with anti-mouse CD45, CD11c, and CD11b antibodies. In these experiments BALF was collected at Days 1, 3 and 8 for comparison to the naïve (saline control) group. Quantitative results are shown for day 1 and 3, where day 3 was observed to be the optimal response to LPS. Note the selective increase in the α7lin+ (YFP+) cell percentage after LPS exposure. (B, C) Cells of the interstitium were isolated from the remaining lung tissues by enzymatic digestion and stained with anti-mouse CD45, CD11b, EpCAM, and CD29. Flow cytometric gating was on either CD45+ cells (B) or on CD45- cells (C). The increase in percentage of CD45- α7lin+ (YFP+) cell after LPS exposure in this time frame was less in comparison to the CD45+ population. (D) Interstitial lung CD45+ and CD45- cells were collected 72 hrs post-challenge with either i.n. saline or LPS using CD45 microbeads and autoMacs sorting. RNA was isolated from these populations (CD45+ or CD45-). In both populations an increase in α7 RNA was observed in response to LPS administration. Combined analysis of α7 gene transcripts from three independent experiments is shown. Data are presented as mean ± SEM.
Mentions: As observed in Fig. 1, naive α7lin+ and α7lin- cells exhibit similar marker expression (also see [28]). To determine if the α7lin+ or α7lin- differ in response to an inflammatory challenge, the response induced by intranasal administration of LPS was measured both in the blood and the different lung compartments (Fig. 2). Animal groups with similar initial α7lin+:α7lin- cell ratios as measured in the blood were administered either saline (i.n.) or LPS (i.n.) and the lungs harvested 24 hrs to 30 days later. We found that in three independent experiments the maximal response to LPS occurs at 72 hrs (Day 3) post LPS challenge (data not shown). Studies [18] using intratracheal administration of LPS report that the peak response in terms of cellular influx occurs at 48 hours which is likely due to the more direct, but more invasive, LPS challenge. We also found that the ratio of α7lin+:α7lin- cells present in BALF or interstitium of unstimulated animals (prior to any treatment) is less than measured in the blood. For example 18% of the cells in the blood are α7lin+ but in the BALF 12% are α7lin+ (Fig. 2A). However, the α7lin+/lin- ratio begins to increase to a statistically significant value relative to the saline control in both cells of the BALF and interstitium by 72 hours after LPS stimulation (Fig. 2A; p < 0.03). By day 8 the α7lin+:α7lin- ratio in both lung compartments returned to near pre-LPS levels where it remains to day 30 (Fig. 2A, B and data not shown). The lung samples from control (saline treated) mice were harvested at 72 hrs, and we observed no influx of inflammatory cells nor an increase in percentage of α7lin+ cells in the lungs at this time or at any of the saline treated points tested (Fig. 2 and not shown). YFP expression by the non-hematopoietic (CD45-) cells was also measured (Fig. 2C). There is a modest increase in the overall CD45- α7lin+:α7lin- ratio at 72 hours following LPS insult.

Bottom Line: This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n.LPS is significantly impaired.Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.

View Article: PubMed Central - PubMed

Affiliation: Geriatric Research, Education and Clinical Center (GRECC), Veterans Affairs Medical Center, Salt Lake City, Utah, United States of America; Department of Internal Medicine, Division of Geriatrics, University of Utah School of Medicine, Salt Lake City, Utah, United States of America; Department of Pathology, Division of Microbiology and Immunology, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

ABSTRACT
The nicotinic acetylcholine receptor alpha7 (α7) is expressed by neuronal and non-neuronal cells throughout the body. We examined the mechanisms of the lung inflammatory response to intranasal (i.n.) lipopolysaccharide (LPS) regulated by α7. This was done in mice using homologous recombination to introduce a point mutation in the α7 receptor that replaces the glutamate residue 260 that lines the pore with alanine (α7E260A), which has been implicated in controlling the exceptional calcium ion conductance of this receptor. The α7E260A mice exhibit normal inflammatory cell recruitment to the blood in response to i.n. LPS administration. This differs from the α7knock-out (α7KO) in which upstream signaling to initiate the recruitment to the blood following i.n. LPS is significantly impaired. While hematopoietic cells are recruited to the bloodstream in the α7E260A mouse, they fail to be recruited efficiently into both the interstitium and alveolar spaces of the lung. Bone marrow reconstitution experiments demonstrate that the responsiveness of both CD45+ and CD45- cells of the α7E260A mouse are impaired. The expression of several pro-inflammatory cytokine and chemokine RNAs including TNFα, IL-1α, Ccl2 and Cxcl10 are decreased in the α7E260A mouse. However, there is a substantial increase in IL-13 expression by CD45- lung interstitial cells in the α7E260A mouse. Our results support the conclusion that α7 functional pleiotropy contributes to modulating the tissue response to an inflammatory insult through impacting upon a variety of mechanisms reflecting the individual cell composition of the lung.

No MeSH data available.


Related in: MedlinePlus