Requirement of Gαi1 and Gαi3 in interleukin-4-induced signaling, macrophage M2 polarization and allergic asthma response

Abstract

IL-4 induces Akt activation in macrophages, required for full M2 (alternative) polarization. We examined the roles of Gαi1 and Gαi3 in M2 polarization using multiple genetic methods. Methods and Results: In MEFs and primary murine BMDMs, Gαi1/3 shRNA, knockout or dominant negative mutations attenuated IL-4-induced IL4Rα endocytosis, Gab1 recruitment as well as Akt activation, leaving STAT6 signaling unaffected. Following IL-4 stimulation, Gαi1/3 proteins associated with the intracellular domain of IL-4Rα and the APPL1 adaptor, to mediate IL-4Rα endosomal traffic and Gab1-Akt activation in BMDMs. In contrast, gene silencing of Gαi1/3 with shRNA or knockout resulted in BMDMs that were refractory to IL-4-induced M2 polarization. Conversely, Gαi1/3-overexpressed BMDMs displayed preferred M2 response with IL-4 stimulation. In primary human macrophages IL-4-induced Akt activation and Th2 genes expression were inhibited with Gαi1/3 silencing, but augmented with Gαi1/3 overexpression. In Gαi1/3 double knockout (DKO) mice, M2 polarization, by injection of IL-4 complex or chitin, was potently inhibited. Moreover, in a murine model of asthma, ovalbumin-induced airway inflammation and hyperresponsiveness were largely impaired in Gαi1/3 DKO mice. Conclusion: These findings highlight novel and essential roles for Gαi1/3 in regulating IL-4-induced signaling, macrophage M2 polarization and allergic asthma response.

Keywords: Gαi1/3; IL-4; M2 polarization; allergic asthma response; signaling.

Figure 1

Gαi1/3 knockdown inhibits IL-4-induced Akt activation and M2 polarization in BMDMs. Primary cultured murine bone marrow-derived macrophages (BMDMs) were treated with IL-4 (100 ng/mL) for 5 min, IL-4Rα, Gαi1, Gαi3 and Gab1 association was tested by co-immunoprecipitation assay (A); Stable BMDMs, expressing the scramble control shRNA (“sh-C”), Gαi1 shRNA and/or Gαi3 shRNA, were treated with IL-4 (100 ng/mL) for applied time, and were tested by Western blotting of listed proteins (B); Relative expression of listed genes (24h after IL-4 treatment) was shown (C-G, and I); The urea production was also tested (H); Cell viability was tested by CCK-8 assay (J). For qPCR, Urea production and viability assays, in each experiment, n=5 (five replicated wells/dishes). Blotting quantification was performed from five replicate blot data (n=5, same for the blotting data in all Figures). Experiments were repeated three times (Same for all following Figures), data of all repeated experiments were pulled together to calculate mean ±SD (Same for all following Figures). “Ctrl” stands for untreated control. *P < 0.01 vs. “Ctrl” treatment in “sh-C” cells (B-I). ^#P < 0.01 vs. IL-4 treatment in “sh-C” cells (B-H). ^&P < 0.01 (B-G).

Figure 2

Gαi1/3 association with APPL1 mediates IL-4Rα internalization and endosomal traffic, essential for IL-4-induced Gab1-Akt-mTOR signaling transduction and M2 responses in macrophages. Primary cultured murine bone marrow-derived macrophages (BMDMs) were treated with IL-4 (100 ng/mL) for 5 min, confocal images were taken to demonstrate the locations of IL-4Rα, EEA1 (the early endosome marker) and DAPI (the nuclear marker) (A); Stable BMDMs, expressing the scramble control shRNA (“sh-C”), Gαi1 shRNA and Gαi3 shRNA (“sh-Gαi1/3”), were treated with or without IL-4 (100 ng/mL) for applied time periods, and tested by Western blotting of listed proteins in total endosomal fractions (B). Stable BMDMs, expressing sh-C or APPL1 shRNA (“sh-APPL1”), were treated with or without IL-4 (100 ng/mL) for listed time periods, listed proteins in total cell lysates (C) and endosomal fractions (D) were tested. Twenty-four hours after IL-4 treatment, relative expression of listed genes (mRNAs and proteins) was shown (E); The cell viability was tested as well (E). Stable BMDMs, with the lentiviral APPL1 construct (“OE-APPL1”) or empty vector (“Vector”), were treated with IL-4 (100 ng/mL) for 10 min, and were tested by Western blotting of listed proteins (F); BMDMs with the empty vector (“Vector”) or the dominant negative Gαi1 construct plus dominant negative Gαi3 construct (“dn-Gαi1/3”), were treated with IL-4 (100 ng/mL), IL-4Rα-APPL1-Gαi1/3 association was tested by co-immunoprecipitation (“IP: IL-4Rα”) (G); Expression of listed proteins, in total cell lysates (“Total”), plasma surface (“Surface”) and endosomal fractions, were tested by Western blotting assays (H-J). Twenty-four hours after IL-4 treatment, relative expression of listed genes (mRNAs and proteins) was shown (K), with cell viability tested as well (L). ***P < 0.001, **P < 0.01.Scale bar=25 µm (A).

Figure 3

Gαi1/3 bind to the intracellular domain of IL-4Rα. Expression of IL-4Rα and Tubulin in the parental control BMDMs (“C”), the stable BMDMs with CRISPR-Cas9-IL-4Rα-KO construct (“IL-4Rα-KO”) or control vector (“Cas9-C”) was shown (A). The IL-4Rα-KO BMDMs were further infected with lentiviral wild-type IL-4Rα construct (Flag-tagged, “IL-4Rα-WT-Flag”) or the lentiviral intracellular domain-depleted IL-4Rα construct (Flag-tagged, “IL-4Rα-ΔIC-Flag”), subjected to puromycin selection to establish stable BMDMs; Established BMDMs were further treated with IL-4 (100 ng/mL) for applied time, and tested by Co-IP (“IP: Flag”) to examine IL-4Rα-APPL1-Gαi1/3 association (B), with expression of listed proteins in total cell lysates tested by Western blotting assays (C). Twenty-four hours after IL-4 treatment, relative expression of listed genes (mRNAs and proteins) was shown (D and E), with the cell viability tested as well (E). The scramble control shRNA (“sh-C”)-expressing BMDMs or Gαi1 shRNA plus Gαi3 shRNA (“sh-Gαi1/3”)-expressing BMDMs (with or without the constitutively-active Akt1 adenovirus [“+Ad-caAkt1”, with green star marker]) were treated with IL-4 (100 ng/mL) for 10 min, tested by immunoblotting of listed proteins (F, Akt phosphorylation was quantified); Relative expression of listed genes (24h after IL-4 treatment) was shown (G); The sh-C BMDMs or Akt1/2-shRNA-expressing stable BMDMs (with or without sh-Gαi1/3) were treated with IL-4 (100 ng/mL) for 10 min, tested by immunoblotting of listed proteins (H, Akt phosphorylation was quantified); Relative expression of listed genes (24h after IL-4 treatment) was shown (I); Stable BMDMs, with the adenovirus Gαi1 construct plus the adenovirus Gαi3 construct (“OE-Gαi1/3”) or empty vector (“Vec”), were treated with IL-4 (100 ng/mL) for applied time, and were tested by Western blotting of listed proteins (J, results were quantified); Relative expression of listed genes (mRNAs and proteins) was tested by qPCR assays (K and M); The urea production was also tested (L), with cell viability tested by CCK-8 (N).“Ctrl” stands for untreated control BMDMs. ***P < 0.001, **P < 0.01.

Figure 4

Gαi1 and Gαi3 DKO inhibits IL-4-induced Akt activation and M2 polarization in BMDMs. BMDMs, derived from both WT mice and Gαi1 and Gαi3 DKO mice (five week old), were treated with IL-4 (100 ng/mL) for applied time, and were tested by Western blotting of listed proteins (A and B); Expression of listed genes (mRNAs and proteins) was tested by qPCR (C-G, I); The urea production was also tested (H), with cell viability tested byCCK-8 assay (J). “Ctrl” stands for untreated control BMDMs. *P < 0.001 (B). *P < 0.001 vs. “Ctrl” treatment in “WT” DMEMs (C-I). ^#P < 0.001 vs. IL-4 treatment in “WT” DMEMs (C-H).

Figure 5

Impaired M2 polarization in human macrophages and Gαi1/3 DKO mice. Human monocytes-derived macrophages (MDMs) were transduced with the lentiviral scramble control shRNA (“sh-C”) or the lentiviral Gαi1 shRNA plus Gαi3 shRNA (“sh-Gαi1/3”) (A and B), the adenovirus Gαi1 construct plus the adenovirus Gαi3 construct (“OE-Gαi1/3”) or empty vector (“Vec”) (C and D), MDMs were then treated with or without IL-4 (100 ng/mL) for 10 min, and tested by Western blotting of listed proteins (A and C). Twenty-four hours after IL-4 treatment, relative expression of listed genes was shown (B and D); (E) M2 genes (Fizz1, Mgl2, IL-10 and Mgl1) expression and Akt activation in peritoneal exudate cells (PECs) from WT and Gαi1/3 DKO mice four days post intraperitoneal (IP) injection with IL-4 complex on days 0 and 2. (F) Relative expression of M2 genes (Fizz1, Mgl2, IL-10 and Mgl1, mRNAs and proteins) as well as Akt activation in PECs from WT and Gαi1/3 DKO mice 48h after IP injection with chitin. In each experiment, n=5 (five replicated wells/dishes). Experiments were repeated five times (five mice per group), data of all repeated experiments were pulled together to calculate mean ±SD. ***P < 0.001, **P < 0.01.

Figure 6

Ovalbumin-induced airway inflammation and hyperresponsiveness are largely impaired in Gαi1/3 DKO mice. WT or Gαi1/3 DKO mice (10 mice per group) were first sensitized and then challenged by OVA or PBS for three days. The airway responsiveness to intravenous acetylcholine chloride (Ach) administration was determined (A); The number of bronchoalveolar lavage (BAL) fluids eosinophils (B), serum total IgE contents (C), serum total OVA-specific IgG1 (D) and IL-4 contents in BAL fluids (E) were determined. Lungs were also fixed and subjected to HE staining and Masson staining (F). Scale Bar= 50 µm. Alveolar macrophages (AMs) were isolated and relative expression of listed genes was tested by qPCR, Western blotting and immunofluorescence assays (G). * P < 0.05 vs. PBS treatment in WT mice (A-E). ^#P < 0.05 vs. OVA treatment in WT mice (A-E). ***P < 0.001 (G), **P < 0.01 (G). Scale bar= 100 µm (G).