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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

The response of α7G, α7E260A:G heterozygote and α7E260A:G homozygote mice to i.n. LPS challenge.Mice (α7G, heterozygote α7E260A+/-:G or homozygote α7E260A+/+:G) were challenged with i.n. LPS (250 μg/mouse in 30 μl Saline) or saline (30 μl per mouse). For all experiments the results are shown as scatter plots and quantitated with bar graphs reflecting the results of three independent experiments (4 to 5 mice per experimental group). Error bars represent standard error of the mean (SEM) and statistical significance was determined using the Student’s t-test. (A) a small sample of blood (50 μl) was collected 24 hours LPS post-challenge and assessed for CD11b+Gr1+ cells (left panel). On day 3 after challenge, mice were sacrificed and BALF and interstitial cells isolated as described above and in the Methods for determination of total number of nucleated cells (A, middle and right panels). (B) BALF cells and (C) interstitial CD45+ cells isolated at 72 hrs post-challenge from α7G, α7E260A+/-:G, α7E260A+/+:G. (D) BALF and interstitial CD45+ cells are shown and further identified by markers for monocyte/macrophages (Ly6C) and PMN (Ly6G).
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pone.0121128.g004: The response of α7G, α7E260A:G heterozygote and α7E260A:G homozygote mice to i.n. LPS challenge.Mice (α7G, heterozygote α7E260A+/-:G or homozygote α7E260A+/+:G) were challenged with i.n. LPS (250 μg/mouse in 30 μl Saline) or saline (30 μl per mouse). For all experiments the results are shown as scatter plots and quantitated with bar graphs reflecting the results of three independent experiments (4 to 5 mice per experimental group). Error bars represent standard error of the mean (SEM) and statistical significance was determined using the Student’s t-test. (A) a small sample of blood (50 μl) was collected 24 hours LPS post-challenge and assessed for CD11b+Gr1+ cells (left panel). On day 3 after challenge, mice were sacrificed and BALF and interstitial cells isolated as described above and in the Methods for determination of total number of nucleated cells (A, middle and right panels). (B) BALF cells and (C) interstitial CD45+ cells isolated at 72 hrs post-challenge from α7G, α7E260A+/-:G, α7E260A+/+:G. (D) BALF and interstitial CD45+ cells are shown and further identified by markers for monocyte/macrophages (Ly6C) and PMN (Ly6G).

Mentions: We next determined the effect of i.n. LPS induced lung inflammatory response in the α7E260A mouse. Each experiment described was performed at least 3 times and n = 4 to 5 mice individually measured per experiment for BALF and interstitial cells. The normal CD45+ cellular composition in the BALF (CD11c+) and lung interstitium (CD11b+) of naive α7E260A:G homozygote and heterozygote mice is equivalent to naive α7G (control) mice (data not shown). Mice (α7G and α7E260A:G) were administered i.n. LPS and a small blood sample was obtained 24 hrs later to determine if a systemic response occurred as determined by the influx of inflammatory cells into the blood. In a non-LPS treated mouse, the percent of Gr1+CD11b+ cells in the blood is typically between 7% and 15% of the nucleated cells (Fig. 4A, left panel). At 24 hrs after i.n. LPS, blood levels of Gr1+CD11b+ cells in all mice increased to 30% or more with no statistically significant difference between mouse strains (α7G to α7E260A:G). This result demonstrates that signaling after the i.n. LPS assault to initiate migration of inflammatory cells is normal in the α7E260A:G mouse. However, lungs harvested at 72 hrs post-LPS administration had significantly fewer α7E260A:G nucleated cells in both the BALF and interstitium when compared to the control α7G (Fig. 4 A, right panels). Significantly fewer Gr1+CD11b+ cells migrated into the BALF and interstitium of LPS challenged α7E260A:G mice compared with α7G animals (Fig. 4B, C). Of the cells in the BALF and interstitium that are affected, Ly6C+Ly6G- cells (monocyte/macrophages) and Ly6C-Ly6G+ (neutrophils) are similarly reduced in both the BALF and interstitium of the α7E260A:G mice (Fig. 4D). Thus, the point mutation associated with α7 calcium permeability significantly reduces the inflammatory cell migration (macrophages and PMNs) from the blood to the lungs of mice exposed to LPS.


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)

The response of α7G, α7E260A:G heterozygote and α7E260A:G homozygote mice to i.n. LPS challenge.Mice (α7G, heterozygote α7E260A+/-:G or homozygote α7E260A+/+:G) were challenged with i.n. LPS (250 μg/mouse in 30 μl Saline) or saline (30 μl per mouse). For all experiments the results are shown as scatter plots and quantitated with bar graphs reflecting the results of three independent experiments (4 to 5 mice per experimental group). Error bars represent standard error of the mean (SEM) and statistical significance was determined using the Student’s t-test. (A) a small sample of blood (50 μl) was collected 24 hours LPS post-challenge and assessed for CD11b+Gr1+ cells (left panel). On day 3 after challenge, mice were sacrificed and BALF and interstitial cells isolated as described above and in the Methods for determination of total number of nucleated cells (A, middle and right panels). (B) BALF cells and (C) interstitial CD45+ cells isolated at 72 hrs post-challenge from α7G, α7E260A+/-:G, α7E260A+/+:G. (D) BALF and interstitial CD45+ cells are shown and further identified by markers for monocyte/macrophages (Ly6C) and PMN (Ly6G).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4372581&req=5

pone.0121128.g004: The response of α7G, α7E260A:G heterozygote and α7E260A:G homozygote mice to i.n. LPS challenge.Mice (α7G, heterozygote α7E260A+/-:G or homozygote α7E260A+/+:G) were challenged with i.n. LPS (250 μg/mouse in 30 μl Saline) or saline (30 μl per mouse). For all experiments the results are shown as scatter plots and quantitated with bar graphs reflecting the results of three independent experiments (4 to 5 mice per experimental group). Error bars represent standard error of the mean (SEM) and statistical significance was determined using the Student’s t-test. (A) a small sample of blood (50 μl) was collected 24 hours LPS post-challenge and assessed for CD11b+Gr1+ cells (left panel). On day 3 after challenge, mice were sacrificed and BALF and interstitial cells isolated as described above and in the Methods for determination of total number of nucleated cells (A, middle and right panels). (B) BALF cells and (C) interstitial CD45+ cells isolated at 72 hrs post-challenge from α7G, α7E260A+/-:G, α7E260A+/+:G. (D) BALF and interstitial CD45+ cells are shown and further identified by markers for monocyte/macrophages (Ly6C) and PMN (Ly6G).
Mentions: We next determined the effect of i.n. LPS induced lung inflammatory response in the α7E260A mouse. Each experiment described was performed at least 3 times and n = 4 to 5 mice individually measured per experiment for BALF and interstitial cells. The normal CD45+ cellular composition in the BALF (CD11c+) and lung interstitium (CD11b+) of naive α7E260A:G homozygote and heterozygote mice is equivalent to naive α7G (control) mice (data not shown). Mice (α7G and α7E260A:G) were administered i.n. LPS and a small blood sample was obtained 24 hrs later to determine if a systemic response occurred as determined by the influx of inflammatory cells into the blood. In a non-LPS treated mouse, the percent of Gr1+CD11b+ cells in the blood is typically between 7% and 15% of the nucleated cells (Fig. 4A, left panel). At 24 hrs after i.n. LPS, blood levels of Gr1+CD11b+ cells in all mice increased to 30% or more with no statistically significant difference between mouse strains (α7G to α7E260A:G). This result demonstrates that signaling after the i.n. LPS assault to initiate migration of inflammatory cells is normal in the α7E260A:G mouse. However, lungs harvested at 72 hrs post-LPS administration had significantly fewer α7E260A:G nucleated cells in both the BALF and interstitium when compared to the control α7G (Fig. 4 A, right panels). Significantly fewer Gr1+CD11b+ cells migrated into the BALF and interstitium of LPS challenged α7E260A:G mice compared with α7G animals (Fig. 4B, C). Of the cells in the BALF and interstitium that are affected, Ly6C+Ly6G- cells (monocyte/macrophages) and Ly6C-Ly6G+ (neutrophils) are similarly reduced in both the BALF and interstitium of the α7E260A:G mice (Fig. 4D). Thus, the point mutation associated with α7 calcium permeability significantly reduces the inflammatory cell migration (macrophages and PMNs) from the blood to the lungs of mice exposed to LPS.

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