It has been documented that CsA reduces the immunopathological damage and facilitates the survival of IAV-infected mice (17). We further investigated the role of CsA in IAV-induced inflammatory responses in mouse lungs. An influenza A/WSN/33 (H1N1)-infected mouse model was established ( Figure 1A ). Mice were injected intraperitoneally with CsA for 4 hours (h) before infected intranasally with IAV. The solution with 2% DMSO was as a negative control. The same treatment of CsA was administered at day 2 post infection. The mRNA and protein levels of pro-inflammation cytokines were determined firstly. CsA reduced the mRNA levels of TNF-α, IL-6, and IL-1β in mouse lungs ( Figure 1B ). Meanwhile, similar results were observed in the levels of these cytokines in bronchoalveolar lavage fluid (BALF) ( Figure 1C ). Then the effects of CsA on the pathogenicity of IAV-infected mice were investigated. After IAV infection, the lung indices (100× lung/body weight) of CsA-pretreated mice were lower than those of control mice ( Figure 1D ). Consistently, a slower weight loss of CsA-pretreated mice was observed compared with that of control mice ( Figure 1E ). In addition, CsA alleviated lung consolidation with less infiltrated inflammatory cells ( Figure 1F ). These results indicate that CsA inhibits IAV-induced inflammatory cytokines production and lung injury in vivo.

Next, the role of CsA in IAV-induced inflammatory responses was examined in bone marrow-derived macrophages (BMDMs) from mice. The results of real-time PCR revealed that CsA inhibited the mRNA levels of TNF-α, IL6, IL1β, CCL2, and CXCL10 in IAV-infected BMDMs ( Figure 1G ). In contrast, the expression of anti-inflammatory cytokines (IL4 and TGF-β) was up-regulated in CsA-treated BMDMs upon IAV infection compared with that in DMSO-treated BMDMs ( Figure 1H ). Taken together, these results indicate that CsA suppresses IAV-triggered inflammatory responses both in vivo and in vitro.

Upon encountering different stimuli, macrophages polarize into M1 macrophages or M2 macrophages, which release a vastly different array of cytokines and chemokines that can either promote or inhibit inflammation (19, 23). We hypothesized that CsA might suppress IAV-triggered inflammatory responses by regulating macrophages polarization. Real-time PCR and western blotting assays were performed to investigate the expression of iNOS (a marker of M1 macrophages) and Arg1 (a marker of M2 macrophages) in IAV-infected BMDMs. As expected, CsA significantly reduced the production of iNOS and enhanced the production of Arg1 ( Figures 2A, B ). Similar effect of CsA on the mRNA and protein expression levels of iNOS and Arg1, as well as the mRNA expression levels of Tnfa and Tgfb1 were observed in IFN-γ-stimulated BMDMs ( Figures 2C, D ) and IL4-stimulated BMDMs ( Figures 2E, F ). These results indicate that CsA is able to regulate macrophages polarization. The flow cytometry was performed to further define the phonotype of BMDMs during IAV infection. As shown in Figure 2G , CsA downregulated the proportion of M1 macrophages and upregulated the proportion of M2 macrophages in IAV-infected BMDMs. Moreover, CsA inhibited IFN-γ-induced M1 polarization ( Figure 2H ) and promoted IL-4-induced M2 polarization in BMDMs ( Figure 2I ). These results suggest that CsA inhibits M1 polarization and promotes M2 polarization in BMDMs.

To determine whether CsA regulated macrophages polarization in mice, the expression levels of iNOS and Arg-1 were further examined in lungs from IAV-infected mice. During IAV infection, the mRNA levels of iNOS ( Figure 3A ) were decreased while those of Arg-1 ( Figure 3B ) were upregulated in CsA-treated mice. In addition, the immunostaining assays were performed. After IAV infection, the F4/80+ and iNOS+ cells were significantly decreased in lungs of CsA-pretreated mice ( Figure 3C ). In contrast, the F4/80+ and Arg1+ cells were increased in lungs of CsA-pretreated mice ( Figure 3D ). Collectively, CsA regulates macrophages polarization in both cell and mouse models.

It is well established that the IFN-γ/STAT1 pathway is critical for M1 polarization and the IL4/STAT6 pathway is necessary for M2 polarization (24). Thus, we examined the effects of CsA on STAT1 and STAT6 phosphorylation upon IAV, IFN-γ, or IL4 stimulation. The immunoblotting results showed that CsA inhibited STAT1 phosphorylation and promoted STAT6 phosphorylation in IAV-infected BMDMs ( Figure 4A ). Furthermore, after stimulation with IFN-γ or IL4, the lower level of pSTAT1 and higher level of pSTAT6 were found in CsA-treated BMDMs ( Figures 4B, C ). Taken together, these results indicate that CsA regulates IFN-γ/STAT1 and IL4/STAT6 signaling pathways to influence macrophages polarization.

It is well known that CypA is the main intracellular receptor of CsA, and many biological roles of CypA can be suppressed by CsA (25, 26). Therefore, we further investigated whether CsA involvement on macrophages polarization requires CypA. When CypA is knocked out, iNOS expression was decreased and Arg-1 expression was increased in IAV-infected ( Figures 5A, B ) or cytokines-stimulated ( Figures 5C, D ) BMDMs. Similar results were also found when WT BMDMs were pretreated with CsA. At the same time, the effect of CsA was diminished in Ppia-knockout (Ppia^-/- ) BMDMs. Moreover, the results of flow cytometry showed that CypA deficiency or CsA treatment led to the decreased proportion of M1 and the increased proportion of M2 in IAV-infected ( Figure 5E ) or cytokines-stimulated ( Figures 5F, G ) BMDMs, and CsA had no influence on macrophages polarization in CypA-deficient BMDMs. We also investigated the effect of CypA on the phosphorylation of STAT1 and STAT6. The lower phosphorylation levels of STAT1 and higher phosphorylation levels of STAT6 were observed in Ppia^-/- BMDMs and CsA-treated WT BMDMs compared with those in WT BMDMs after IAV infection. Meanwhile, CsA had no effect on the phosphorylation of STAT1 and STAT6 in CypA-deficient BMDMs ( Supplementary Figure 1A ). Similar results were observed in IFN-γ-stimulated BMDMs ( Supplementary Figure 1B ) and IL4-stimulated BMDMs ( Supplementary Figure 1C ). Collectively, CypA deficiency decreases the polarization of macrophages into pro-inflammatory M1 phenotype and increases the polarization of macrophages into anti-inflammatory M2 phenotype just as CsA does and CsA regulates BMDMs polarization by targeting CypA to block its isomerase activity.

We further investigate whether the role of CsA in inflammatory responses is dependent on CypA in IAV-induced mice. We used the influenza A/WSN/33 model described on Figure 1A ( Figure 6A ). The results of real-time PCR and ELISA assays revealed that the mRNA and protein levels of inflammatory cytokines in lungs of Ppia^-/- mice or CsA-treated WT mice were lower than those in lungs of WT mice, and the effect of CsA was diminished in Ppia^-/- mice ( Figures 6B, C ). The pathological indices also indicated that the effect of CypA deficiency was similar with that of CsA treatment, and CsA no longer attenuated the pathological injury in Ppia^-/- mice ( Figures 6D –F ). Moreover, the effect of CypA deficiency on Nos2 and Arg1 transcriptional levels in mouse lungs were similar with that of CsA treatment, and CsA no longer had effect on Nos2 and Arg1 expression in Ppia^-/- mice ( Figure 6G ), suggesting that CypA played an important role in regulating macrophages polarization. Taken together, these results indicate that CsA suppresses IAV-induced inflammatory responses by targeting CypA.

Female C57BL/6 mice (6-8 weeks old, 18-22 g) were purchased from Vital River Laboratory Animal Center (Beijing, China). Ppia-knockout (Ppia^-/- ) 129 mice were purchased from Jackson Laboratory and backcrossed for at least six generations to C57BL/6 mice. All of the animals were maintained in a barrier facility with free access to food and water.

The Madin–Darby canine kidney cells (MDCK, ATCC CRL-3216) was obtained from the American Type Culture Collection (ATCC). MDCK cell lines were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Switzerland) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under a humidified atmosphere containing 5% CO₂. BMCs (bone marrow cells) were isolated from aseptically dissected and flushed tibias and femurs of 6-8 weeks old C57BL/6 mice. BMCs were differentiated into bone marrow-derived macrophages (BMDMs) for 5-7 days in 1640 medium (Gibco) with 10% (v/v) FBS, 20 ng/ml macrophage-colony stimulating factor (M-CSF), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under a humidified atmosphere containing 5% CO₂.

Influenza A/WSN/33 (H1N1) was rescued from cDNA (46). Viruses were propagated in 9-day-old specific pathogen-free embryonic eggs or MDCK cells for 36–72 h at 37°C, then the allantoic fluid and supernatant was harvested, centrifuged, and stored at -80°C.

For flow cytometry, F4/80-PE, CD80-Pacific Blue and CD206-APC antibodies were obtained from Thermo Fisher. For western blotting assays, the following antibodies were used: anti-pSTAT1 (1:1000, 9167S, CST), anti-STAT1 (1:500, sc-464, Santa Cruz), anti-pSTAT6 (1:1000, 56554S, CST), anti-STAT6 (1:1000, 5397S, CST), anti-GAPDH (1:2000, ab8245, Abcam). For immunostaining assays, the following antibodies were used: anti-F4/80 (1:100, 30325S, CST), anti-iNOS (1:100, 32027S, CST), anti-Arg1 (1:100, 93668S, CST). The CsA powder (S2286) was purchased from Selleck. For assays in vitro, the powder was dissolved with DMSO and 0.1% DMSO was the final concentration for cell culture. For assays in vivo, the powder was dissolved in a solution of 2% DMSO, 30% PEG300, and 5% Tween 80 in sterile ddH₂O.

MDCK were inoculated with 10-fold serially diluted IAV and incubated at 37°C with 5% CO₂ for 2 h. The supernatant was removed; cells were washed with PBS, and then overlaid with DMEM containing 0.6% low-melting-point agarose and 1 μg/mL L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin. After incubation for 48-72 hour, the virus titers were determined by visible plaques.

BMDMs were infected with IAV (MOI = 1) in DMEM containing 0.5 μg/mL TPCK-treated trypsin for 2 h. Subsequently, the supernatant was removed; cells were washed with PBS and overlaid with infection media (DMEM with 100 U/mL penicillin and 100 μg/mL streptomycin). Cells were collected at indicated time for the subsequent real-time PCR, flow cytometry and western blotting.

BMDMs were polarized in 1640 medium containing IFN-γ (20 ng/mL) or IL4 (20 ng/mL), 100 U/mL penicillin and 100 μg/mL streptomycin for indicated time, followed by collected and used for real-time PCR, flow cytometry and western blotting.

6–8 weeks old C57BL/6 mice were anesthetized and inoculated intranasally with IAV (5000 PFU) in 30 μL of PBS. After 1 or 3 day, the mice were sacrificed and the lungs were used in subsequent real-time PCR, lung index analysis, Hematoxylin and eosin (H & E) staining and immunostaining assays. Mouse body weights were monitored.

Total RNA was extracted from BMDMs and lung tissue homogenate with TRIzol (Invitrogen) according to the manufacturer’s instructions. The cDNA was synthesized from 1-2 μg of total RNA using an oligo (dT) primer and M-MLV reverse transcriptase (Promega) according to the manufacturer’s instructions. The relative gene expression was analyzed by real-time PCR that using TB Green premix (TaKaRa). Primers used in this study: Tnfa, forward primer: 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′, and reverse: 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′; Il6, forward primer: 5′-CCA GAA ACC GCT ATG AAG TTC C-3′, and reverse: 5′-TTG TCA CCA GCA TCA GTC CC-3′; Il1b, forward primer: 5′-GTG GCT GTG GAG AAG CTG TG-3′, and reverse: 5′-GAA GGT CCA CGG GAA AGA CAC-3′; Ccl2, forward primer: 5′-CAG CCA GAT GCA ATC AAT GCC-3′, and reverse: 5′-TGG AAT CCT GAA CCC ACT TCT-3′; Cxcl10, forward primer: 5′-CCA AGT GCT GCC GTC ATT TTC T-3′, and reverse: 5′-TTC CCT ATG GCC CTC ATT CTC A-3′; Il4, forward primer: 5′-CCC CAG CTA GTT GTC ATC CTG-3′, and reverse: 5′-CAA GTG ATT TTT GTC GCA TCC G-3′; Il10, forward primer: 5′-CTT TAA GGG TTA CTT GGG TTG CC-3′, and reverse: 5′-TCC TGA GGG TCT TCA GCT TCT CA-3′; Tgfb1, forward primer: 5′-TTA GGA AGG ACC TGG GTT GGA-3′, and reverse: 5′-CCG GGT TGT GTT GGT TGT AGA-3′; Nos2, forward primer: 5′-GGC AGC CTG TGA GAC CTT TG-3′, and reverse: 5′-GCA TTG GAA GTG AAG CGT TTC-3′; Arg1, forward primer: 5′-CTC CAA GCC AAA GTC CTT AGA G-3′, and reverse: 5′-AGG AGC TGT CAT TAG GGA CAT C-3′; Gapdh, forward primer: 5′-GGT GGT CTC CTC TGA CTT CAA CA-3′, and reverse: 5′-GTT GCT GTA GCC AAA TTC GTT GT-3′. The Ct values generated from ABI 7500 and were analyzed by 2^−ΔΔCt method. The expression of target genes was normalized to Gapdh.

Cells were lysed in lysis buffer (150 mM NaCl, 20 mM Hepes, 1 mM EDTA, 1% TritonX-100, 10% glycerin and protease or phosphatase inhibitors cocktail). The total protein was quantified by BCA protein assay kit (Thermo Fisher) and the same mass of protein samples with 5×Loading buffer were loaded on 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then electroblotted onto a 0.45 μm PVDF membrane. The membranes were blocked by TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 8.0) with 5% (w/v) non-fat dry milk and 1% (w/v) BSA for 1.5 h, followed by incubated with primary antibodies at room temperature for 2 h and washed by TBST. Subsequently, the HRP conjugated specific secondary antibodies was used to bind target primary antibodies for 1 h. After washing by TBST, the target bands were visible by using the ECL western blotting substrate. The expression of target protein was normalized to GAPDH.

Bronchoalveolar lavage fluid (BALF) was obtained from the lungs of mice by washing with 1 mL PBS and collected after centrifugation. The concentration of TNF-α (MTA00B, R&D systems), IL6 (M6000B, R&D systems) and IL1β (MLB00C, R&D systems) in BALF were measured by ELISA according to the manufacturer’s instructions.

FACS analyses were done on BD FACSAria. Data were analyzed with the FlowJo software (Tree Star Inc.).

The severity of the lung injury was analyzed in a blinded manner with the grader unaware of the concrete group being reviewed. The lung histopathological changes were assessed by the four identifiable pathologic processes: (1) alveolar congestion, (2) hemorrhage, (3) leukocyte infiltration or aggregation of neutrophils in airspace or the vessel wall, and (4) thickness of the alveolar wall. The scores of 0 to 4 were defined to represent normal lungs, lower than 25%, 25–50%, 50–75%, and higher than 75% lung involvement, respectively.