Actin fence therapy with exogenous V12Rac1 protects against acute lung injury
Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya
Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya
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Research Article Pulmonology

Actin fence therapy with exogenous V12Rac1 protects against acute lung injury

Abstract

High mortality in acute lung injury (ALI) results from sustained proinflammatory signaling by alveolar receptors, such as TNF-α receptor type 1 (TNFR1). Factors that determine the sustained signaling are not known. Unexpectedly, optical imaging of live alveoli revealed a major TNF-α–induced surge of alveolar TNFR1 due to a Ca2+-dependent mechanism that decreased the cortical actin fence. Mouse mortality due to inhaled LPS was associated with cofilin activation, actin loss, and the TNFR1 surge. The constitutively active form of the GTPase, Rac1 (V12Rac1), given intranasally (i.n.) as a noncovalent construct with a cell-permeable peptide, enhanced alveolar filamentous actin (F-actin) and blocked the TNFR1 surge. V12Rac1 also protected against ALI-induced mortality resulting from i.n. instillation of LPS or of Pseudomonas aeruginosa. We propose a potentially new therapeutic paradigm in which actin enhancement by exogenous Rac1 strengthens the alveolar actin fence, protecting against proinflammatory receptor hyperexpression, and therefore blocking ALI.

Authors

Galina A. Gusarova, Shonit R. Das, Mohammad N. Islam, Kristin Westphalen, Guangchun Jin, Igor O. Shmarakov, Li Li, Sunita Bhattacharya, Jahar Bhattacharya

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Figure 2

Calcineurin-dependent alveolar TNFR1 expression.

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Calcineurin-dependent alveolar TNFR1 expression. (A) Alveoli were optica...
(A) Alveoli were optically imaged to quantify fluorescence of F-actin (Lifeact) and TNFR1 (immunofluorescence) in WT and calcineurin-Aβ–null (CnAβ–/–) mice. Data were obtained as whole-image fluorescence (gray levels above background) at baseline (dashed line) and 30 minutes after alveolar microinfusion of TNF-α. Microinjections of anti-TNFR1 Ab for TNFR1 detection were given prior to and 30 minutes after TNF-α. Data are presented as ± SEM. n = 5 lungs for each group. *P < 0.05 compared with baseline using ANOVA with Bonferroni correction. (B) Immunoblots and densitometry of lung lysates obtained 4 hours after i.n. TNF-α. Replicated 3 times. *P < 0.05 as indicated. (C and D) Cofilin transfections were for WT plasmid (pWT), and for constitutively active (pS3A), or inactive (pS3E) cofilin mutants. LatB, latrunculin B. Images in C show alveolar F-actin in terms of rhodamine-phalloidin fluorescence at baseline (upper panel) and 30 minutes after alveolar injection of TNF-α (lower panel). The images in D were obtained at baseline after the first microinjection of anti-TNFR1 Ab (upper panel). A second Ab microinjection for TNFR1 detection was given 30 minutes after TNF-α microinjection (lower panel). The data in C and D are quantifications of whole-image fluorescence (mean ± SE). Alv, alveolus. Scale bars: 50 μm. Each dot shows data for a single lung. n = 5 for all groups, except LatB (n = 3 for pWT and pS3A, n = 4 for pS3E). *P < 0.05 versus baseline for the same group using 2-tailed t test; #P < 0.05 baseline for the pWT group using ANOVA with Bonferroni correction.