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

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.


Ethics
Protocols of this study were approved by the Ethics Committee of Soochow University.

Murine BMDMs
The bone marrow of WT and Gαi1/3 DKO mice [15,16] were flushed by complete RPMI medium (with FBS), with the resulting cell pellets resuspended in ACK hypotonic buffer. The remaining bone marrow cells were washed with complete RPMI medium, and cultured in RPMI medium with 30% L-929 cell medium [22]. Within 8-10 days the adherent primary bone marrow-derived macrophages (BMDMs) were trypsinized, washed and re-plated for the further experimental usage.

Gαi1/3 shRNA
At 100, 000 cells per well, MEFs or BMDMs were seeded into six-well tissue culture plates, and Gαi1 shRNA lentivirus and/or the Gαi3 shRNA lentivirus [15,16] were added. The culture medium was replaced with fresh puromycin-containing culture medium every two days, until resistant colonies were formed (10-12 days). In stable cells Gαi1/3 knockdown (over 90% knockdown efficiency) was verified by Western blotting and quantitative real-time PCR (qPCR).

CRISPR/Cas9 knockout of Gαi1 and Gαi3
The lentiviral CRISPR/Cas-9 Gαi1 KO construct and lentiviral CRISPR/Cas-9 Gαi3 KO construct were designed and purchased from Shanghai Genechem (Shanghai, China) [15], transfected into MEFs/BMDMs, and selected with puromycin. Control cells were treated with the empty vector with control sgRNA (Santa Cruz Biotech). In stable cells Gαi1/3 knockout was confirmed by Western blotting and qPCR.

Gαi1/3 overexpression or mutation
At 100, 000 cells per well, BMDMs or human macrophages were seeded into six-well tissue culture plates, murine/human Gαi1-expressing adenovirus (Ad-Gαi1) and murine Gαi3-expressing adenovirus (Ad-Gαi3) [15,16] were added. Cells were selected by puromycin. Control cells were treated with the empty vector-expressing adenovirus. In stable cells Gαi1/3 overexpression was confirmed by Western blotting and qPCR. The dominate negative Gαi1 construct and the dominate negative Gαi3 construct were described in our previous study, co-transfected to cultured BMDMs [19].

Generation of the Gαi1/3 double knock (DKO) mice
The generation of Gαi1/3 DKO mice by the CRISPR-Cas9 method was described previously [15,16]. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of Soochow University. The protocols was approved by the Committee on the Ethics of Animal Experiments of Soochow University.

qPCR
The detailed protocols for qPCR by the ABI-7600 Prism equipment and the SYBR Green PCR kit were previously described [15,23]. mRNA expression of targeted genes was quantified via the the ΔΔ Ct protocol. Samples from BMDMs were normalized to hypoxanthine phosphoribosyltransferase (HPRT) and samples from PECs were normalized to the macrophage marker CD68. The primers were reported early [14]. Other primers were synthesized and verified by Genechem Co. (Shanghai, China).
Western blotting and data quantification, co-immunoprecipitation (IP) assay, CCK-8 viability assay, and confocal microscopy were described in detail in our previous studies [16,18,19,24]. For all the Western blotting assay, each lane of a SDS-PAGE Gel was loaded with exact same amount of quantified protein lysates (40 μg in each treatment), the same set of lysate samples were run in parallel ("sister") gels to test different proteins (same for all Figures).

Plasma membrane fractionation
The detailed protocols for plasma membrane isolation was described previously [15,16].

Endosome fractions
BMDMs with the applied treatments were harvested and re-suspended in the hypotonic swelling buffer [25], and lysed with 30 strokes in a Dounce homogenizer using a tight pestle, and swelling was stopped by the addition of two fold homogenization buffer [25]. Lysates were centrifuged to obtain the post-nuclear supernatants, which was further centrifuged [25]. The resulting supernatants were centrifuged, and the pellet solubilized in the homogenization buffer [25]. Insoluble particles were removed by short centrifugation and the supernatant loaded onto a 5-20% continuous OptiprepTM (Sigma-Aldrich), poured using homogenization buffer. The gradient was further centrifuged at 60,000 g for 24h, with total 10 endosomal fractions collected, and proteins precipitated with 12% TCA for 1h. Fractions were centrifuged at 12,000 g for 1h. The protein pellets, combining all ten endosomal fractions, were dissolved in SDS-sample buffer for analysis by Western blotting.

APPL1 overexpression or silencing
Briefly, the full-length mouse APPL1 (provided by Genechem, Shanghai, China) was sub-cloned into the GV248 construct, and transfected into human embryonic kidney 293 (HEK-293) cells, together with the lentivirus packaging plasmids (pCMV-VSVG and pCMV-ΔA.9, Genechem). The generated lentivirus was filtered, enriched and added to cultured BMDMs. Stable BMDMs were then established by culturing in the puromycin-containing medium. The murine APPL1 shRNA lentiviral particles (sc-61981-V) were provided by Santa Cruz Biotech (Beijing, China), added to cultured BMDMs (cultured in polybrene medium). Stable BMDMs were again established by puromycin selection. APPL1 expression was always verified by Western blotting assays in the stable BMDMs.

CRISPR/Cas9 knockout of IL-4Rα
The murine IL-4Rα CRISPR/Cas9 KO construct (sc-421111) was purchased from Santa Cruz Biotech (Beijing, China). The plasmid was transfected to BMDMs via Lipofectamine 2000 (Thermo-Fisher, Invitrogen, Shanghai, China). Single BMDMs were cultured for two weeks, subjected to screen of IL-4Rα KO by qPCR and Western blotting assays. Stable BMDMs with complete depleted IL-4Rα were utilized for further experiments.

Chitin administration
As described previously [29], chitin was washed and sonicated on ice. The dissolved chitin was filtered and diluted within PBS to a concentration of 4 μg/mL. For each mouse, 800 ng chitin (dissolved in 200 μL PBS) was injected intraperitoneally, after 48h PECs were collected.

Primary human macrophages
Primary human monocyte-derived macrophages (MDM) were provided by Dr. Sun at Shanghai Pulmonary Hospital [30]. Macrophages were obtained from CD14 magnetic bead-selected monocytes [31] from peripheral blood mononuclear cells (PBMCs) of written-informed consent healthy donors [31]. The detailed protocols for primary human macrophage cultivation were previously described [31].

Ovalbumin-induced mouse asthma model
WT or Gαi1/3 DKO mice were sensitized by intraperitoneal injection of ovalbumin (OVA, twice, one week apart, Sigma) [32]. One week following the last sensitization, mice were anesthetized and challenged with OVA or PBS as described [32]. Airway responsiveness, pulmonary inflammation and immunoglobulin synthesis were compared in wild-type and Gαi1/3 DKO mice sensitized and challenged with PBS or OVA. Three days after aspiration challenge, airway responsiveness to intravenous acetylcholine chloride (Ach) administration was determined using the described protocol [32]. The number of inflammatory cells in bronchoalveolar Lavage (BAL) was determined. Lungs were also fixed and subjected to HE staining and Masson staining. Mouse lung tissues were digested and minced as reported [33]. After lysis of red blood cells (RBCs), the dissociated cells were underlaid with 7.5 mL of lymphocyte separation medium (Sigma, Shanghai, China) and cells were centrifuged. From the middle layer the mononuclear cells were incubated in six-well plates for two hours [33]. Thereafter, the adherent cells were alveolar macrophages.

Statistical analysis
Numerical data and and histograms presented were expressed as means ± standard deviation (SD). Comparison between any two groups was by two-tailed unpaired Student t test. Multiple group comparison was performed by one-way analysis of variance (ANOVA) with post hoc Bonferroni test (data were all with normal distribution). Values of P less than 0.05 were considered statistically significant.

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).
The results were further validated using dominant negative (dn) mutants of Gαi1 and Gαi3 that precluded Gαi1/3 binding to adaptor/associated proteins [18,19] leading to defective M2 responses in BMDMs.

Gαi1 and Gαi3 are required for IL-4-induced Akt activation and Th2 response in human macrophages
In human monocytes-derived macrophages (MDMs), shRNA (see our previous study [15]) was utilized to knockdown both Gαi1 and Gαi3. The applied shRNA lentivirus resulted in significant Gαi1 and Gαi3 protein double downregulation (sh-Gαi1/3, Figure 5A). In line with the results in BMDMs, IL-4-induced phosphorylation of Akt and S6K1 was largely inhibited by sh-Gαi1/3 in MDMs (Figure 5A). In contrast, expression of IL-4Rα and STAT6 as well as IL-4-induced STAT6 phosphorylation were unchanged ( Figure 5A). As there is a significant difference between the transcriptional response toward IL-4 in human and murine macrophages [41,42], we analyzed the potential role of sh-Gαi1/3 on expression of Th2 response genes in MDMs, including CCL17 and CAMK2A [41,42]. The qPCR assay results, Figure 5B, showed that IL-4 significantly increased expression of CCL17 and CAMK2A in MDMs. This response was largely inhibited by sh-Gαi1/3 ( Figure  5B).

Ovalbumin-induced airway inflammation and hyperresponsiveness are largely impaired in Gαi1/3 DKO mice
Lung IL-4 signaling and M2 macrophages are key regulators of airway responses to inhaled allergens, participating in poor lung function in allergic asthma [32,[44][45][46]. We therefore compared the effects of OVA sensitization and challenge on the development of allergic airway responses in WT and Gαi1/3 DKO mice. Assessing airway responsiveness to intravenous acetylcholine chloride (Ach) administration [32], following OVA sensitization and challenge WT mice developed significant increases in airway pressure time index (APTI) after Ach administration ( Figure 6A). In contrast, the airway reactivity was significantly lower in OVA-treated Gαi1/3 DKO mice (P < 0.05 vs. WT mice, Figure 6A), suggesting that Gαi1/3 are involved in OVA-induced airway hyperresponsiveness (AHR). IL-4 plays an important role in eosinophilia by increasing IL-5 production and upregulation of endothelial VCAM-1 expression, to promote attachment and migration of eosinophils [44]. As expected, OVA sensitization and challenge induced a striking increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluids of WT mice (Figure 6B). Although increases in BAL eosinophils were detected OVA-treated Gαi1/3 DKO mice, they were much lower than OVA-treated WT animals ( Figure 6B).
IL-4 is also important for IgE synthesis required for the pathogenesis of allergic responses. We found that OVA-treated WT mice produced a large amount of serum IgE (Figure 6C). OVA-stimulated IgE production was however attenuated in Gαi1/3 DKO mice (P < 0.05 vs. WT mice, Figure 6C). In addition, we examined serum levels of OVA-specific IgG1, a method utilized to assess Th2 cytokines in vivo. Compared to OVA-treated WT mice (Figure 6D), the serum OVA-specific IgG1 levels were inhibited in Gαi1/3 DKO mice (P < 0.05 vs. WT mice, Figure 6D). In OVA-challenged WT mice, IL-4 contents in BAL were significantly increased, but were much lower in the BAL of OVA-challenged DKO mice (see revised Figure 6E). These results further demonstrate inhibition of the Th2 response in OVA-treated Gαi1/3 DKO mice.
Examining pulmonary histopathology with HE and Masson staining demonstrated that in OVA-treated WT mice the bronchial wall was thickened and the lumen was narrow (Figure 6F). A significant amount of mucus was detected in the lumen, with several red mucus plugs observed as well ( Figure 6F). In addition, a large number of inflammatory cells, including lymphocytes, eosinophils, neutrophils, were infiltrated into bronchus and blood vessels ( Figure 6F). In contrast, in the lung of OVA-treated Gαi1/3DKO mice, the bronchioles and alveoli were mainly intact, with few necrotic or exfoliated epithelial cells (Figure 6F). The number of infiltrated inflammatory cells was significantly lower when compared to WT mice ( Figure 6F). These results show that Gαi1/3DKO protects against OVA-induced airway hyperresponsiveness and mucus production. Alveolar macrophages (AMs) were isolated from OVA-treated mice. In AMs of OVA-treated Gαi1/3 DKO mice expression of M2 genes (Arg1, Fizz1, Mgl2, and Mgl1) was significantly lower than that in AMs of OVA-treated WT mice ( Figure 6G). Western blotting and immunofluorescence assay results further confirmed Arg1 and Fizz1 protein levels in AMs of OVA-treated Gαi1/3 DKO mice were significantly lower than those in AMs of OVA-treated WT mice ( Figure 6G). Akt-S6K phosphorylation was inhibited as well ( Figure 6G). These results further suggest that Gαi1/3 are important genes for IL4-induced macrophage M2 polarization and Th2 response in vivo.

Following
IL-4 stimulation, IL-4Rα is internalized to mediate ligand uptake [26]. The intracellular domain of IL-4Rα initiates Rac1-, Pakand actin-mediated endocytosis, leading to an increased receptor density at endosomes [36]. Whether IL-4Rα endocytosis is essential for downstream signaling transduction is still under debate, and could be cell-type-dependent. Kurgonaite et al., showed that in HEK293T cells IL-4Rα endocytosis is required for IL-4-induced JAK/STAT6 activation [36], although Friedrich et al., demonstrated that IL-4Rα endocytosis is not functionally connected to JAK/STAT6 activation in macrophages [26].
Our previous studies show that Gαi1/3 play an essential role in the formation of the VEGFR2 endocytosis complex, required for VEGFR2 endocytosis and downstream signaling activation by VEGF [15]. Furthermore, BDNF-induced TrkB endocytosis and downstream signaling activation are blocked in Gαi1/3-depleted cells and neurons [16]. The results of the present study demonstrate that Gαi1 and Gαi3 physically associate with the intracellular domain of IL-4Rα, which is essential for IL-4Rα endocytosis and Akt activation, but not STAT6 activation. Disruption of IL-4Rα-Gαi1/3 association, by Gαi1/3 silencing/KO, dominant negative mutants, or though deletion of the intracellular domain of IL-4Rα, abrogated IL-4-induced IL-4Rα endocytosis, endosomal traffic, and Akt activation, while leaving STAT6 unaffected. Therefore, in IL-4-treated BMDMs, Gαi1/3-mediated IL-4Rα endocytosis and endosomal traffic are essential for activation of Akt, but not STAT6.
The APPL1 adaptor localized to endosomes serves as a platform for the assembly and trafficking of receptors and endosomal signaling [38,39]. Following IL-4 stimulation, we found that APPL1 association with Gαi1/3 and IL-4Rα is required for IL-4Rα endosomal traffic and Gab1-Akt-mTOR activation in BMDMs. IL-4-induced IL-4Rα endosomal traffic, Gab1-Akt-mTOR activation and M2 response were potently inhibited by APPL1 silencing. Conversely, ectopic overexpression of APPL1 promoted IL-4 signaling. Therefore, we propose that APPL1 is a critical adaptor protein for IL-4-induced M2 signaling.
Our previous studies have confirmed that in RTK signaling, Gαi1/3 are essential for Gab1 activation in response to various growth factors [17][18][19]. In response to IL-4, macrophages can polarize towards M2, leading to expression of multiple M2 biomarkers, including Arg1, Mgl1, Mgl2 and Fizz1. IL-4-induced activation of PI3K-AKT activation is vital in regulating expression of M2 markers [37]. Following IL-4 stimulation, Gab1 preferentially interacted with p85 to activate PI3K-AKT signaling [37]. In the present study, we show that Gab1 recruitment to IL4-activated IL-4Rα and subsequent activation were largely impaired when Gαi1/3 were silenced, depleted, or mutated. In Gab1 KO MEFs treated with IL-4, Akt activation was completely blocked, while Gαi1/3 expression and STAT6 activation were intact. Therefore, Gαi1/3 are required for IL4-induced Gab1 recruitment and activation, which mediates downstream Akt activation and M2 responses. One possibility is that IL-4-induced IL-4Rα endosomal traffic was disrupted with Gαi1/3 silencing, KO or mutation, that should block the docking of the adaptor protein Gab1, thus inhibiting Gab1 activation and downstream p85-Akt activation.
IL-4 and other Th2 cytokines are responsible for recruiting leukocytes to the site of inflammation, essential for IgE synthesis, airway eosinophilia, mucus secretion, and ultimately airway hyperresponsiveness (AHR) [55,56]. Studies have shown that injection or overexpression of IL-4 in the airways could induce airway eosinophilia and AHR [55]. Here, using an asthma mouse model we found that OVA-stimulated IgE production, airway eosinophilia, inflammatory cells infiltration and AHR were largely impaired in Gαi1/3 DKO mice. Furthermore, expression of M2 markers in ex vivo alveolar macrophages-derived from Gαi1/3 DKO mice was significantly lower than that in alveolar macrophages of WT mice. The results of this study suggest that Gαi1/3 could be novel and key mediators of allergic asthma pathogenesis. Targeting Gαi1/3 could provide a new therapeutic modality for allergic asthma patients.

Conclusion
The results of the present study reveal novel and essential roles of Gαi1/3 proteins in the control of IL-4 signaling, macrophage functions and M2 polarization, with broad implications for regulation of Th2 immunity, inflammation, and allergy.