SP-R210 isoforms of Myosin18A modulate endosomal sorting and recognition of influenza A virus infection in macrophages

Abstract

Influenza A virus (IAV) infection causes acute and often lethal inflammation in the lung. The role of macrophages in this adverse inflammation is partially understood. The surfactant protein A receptor 210 (SP-R210) consists of two isoforms, a long (L) SP-R210_L and a short (S) SP-R210_S isoform encoded by alternative splicing of the myosin 18A gene. We reported that disruption of SP-R210_L enhances cytosolic and endosomal antiviral response pathways. Here, we report that SP-R210_L antagonizes type I interferon β (IFNβ), as depletion of SP-R210_L potentiates IFNβ secretion. SP-R210 antibodies enhance and attenuate IFNβ secretion in SP-R210_L replete and deficient macrophages, respectively, indicating that SP-R210 isoform stoichiometry alters macrophage function intrinsically. This reciprocal response is coupled to unopposed and restricted expression of viral genes in control and SP-R210_L-deficient macrophages, respectively. Human monocytic cells with sub-stoichiometric expression of SP-R210_L resist IAV infection, whereas alveolar macrophages with increased abundance of SP-R210_L permit viral gene expression similar to murine macrophages. Uptake and membrane binding studies show that lack of SP-R210 isoforms does not impair IAV binding and internalization. Lack of SP-R210_L, however, results in macropinocytic retention of the virus that depends on both SP-R210_S and interferon-inducible transmembrane protein-3 (IFITM3). Mass spectrometry and Western blot analyses indicate that SP-R210 isoforms modulate differential recruitment of the Rho-family GTPase RAC1 and guanine nucleotide exchange factors. Our study suggests that SP-R210 isoforms modulate RAC-dependent macropinosomal sorting of IAV to discrete endosomal and lysosomal compartments that either permit or prevent endolysosomal escape and inflammatory sensing of viral genomes in macrophages.

Keywords: IFITM3; Influenza life-cycle; Macrophages; Macropinocytosis; Myosin 18A (MYO18A); Surfactant protein A receptor 210 (SP-R210).

Figure 1.. SP-R210_L mediates viral genome expression and suppression of antiviral response in macrophages.

Cells were infected with 2 MOI of IAV strains Phil82 (B,C), PR8 NS1-GFP (B), PR8 (B,C, and D-G), and Aichi (C) for 10-hours and analyzed by flow cytometry utilizing an influenza nuclear protein (NP) antibody or an NS1-GFP reporter strain to monitor uptake and expression of NP or NS1 over time as described in Methods section. (A) Western blotting comparison shows differential expression of SP-R210_L vs. SP-R210_S in human AMs, KG-1 cells and similar expression in RAW264.7 control cell extracts consistent with previous studies [27-29, 37, 48, 115]. (B, C) Expression of the IAV genome is suppressed in SP-R210_L(DN) and KG-1 cells compared to control and human alveolar macrophages, respectively. In (B), NP was monitored at 6 (faint grey histogram), 12 (dashed line histogram), and 24 (black line histogram)-hrs after infection using either an NP antibody (FITC-NP) or the NS1-GFP reporter. In (C) flow cytometry was performed 12-hrs after infection. (D) Disruption of SP-R210_L enhances expression of IFNβ in response to PR-8 infection. IFNβ was measured by ELISA in media harvested over time. Data shown are means±SD from n=2 independent experiments performed in duplicate. **p<0.01 compared to t=0, #p<0.01 compared to compared to control cells at each time point. (E) Polyclonal SP-R210 antibodies targeting the unique carboxy-terminal domain of SP-R210 suppress IAV infection in a concentration-dependent manner. Control and SP-R210_L(DN) cells treated with increasing concentrations of anti-SP-R210 antibody (5, 10, 20, 40, 100, and 200 μg/mL) for 5-hours, upon which cells were washed and 2 MOI IAV was added to the cells in 1:1 DMEM and the appropriate concentration of anti-SP-R210 antibodies or IgG control. Infection was allowed to proceed for a total of 12-hours and then cells were harvested and stained against IAV NP as an indirect measure for IAV uptake and replication. SP-R210 antibody treatment of RAW264.7 cells resulted in a dose-dependent inhibition of IAV infection in macrophages, suggesting that SP-R210 may be involved in the macrophage response to IAV. Data shown are means±SD from N=2 independent experiments performed in duplicate. *p<0.05 compared to 200 μg/mL IgG pre-immune control. (F) SP-R210 antibodies induce and attenuate secretion of IFNβ in response to IAV infection in control and SP-R210_L(DN) cells, respectively. Data shown are means±SD from N=2 independent experiments performed in duplicate. **p<0.01 and *p<0.05 compared to pre-immune IgG, #p<0.01 compared to compared to control cells at respective time points. (G) SP-R210 antibodies enhance secretion of IFNβ in response to IAV infection in in human AMs. Data shown are means±SD from N=2.

Figure 2.. Differential cytokine responses in control and SP-R210-deficient macrophages.

Control, SP-R210_L(DN), and SP-R210(KO) cells cultured overnight in FBS replete DMEM were washed and placed in 0.5 mL of 1:1 ratio of serum-free DMEM to PBS in the presence or absence of 20 μg/mL Poly(I:C) or 5 μg/mL Imiquimod. Media were collected for the 2-hour time point and then replaced with 0.5 mL DMEM/10% FBS and media harvested at 2, 6, and 22-hours. The concentration of IFNβ (A,B) and TNFα (C,D) was quantitated by ELISA. N=3. All data are shown as means±SD. (A) **p<003 and ***p<0.0005 for SP-R210_L(DN) vs SP-R210_L(DN)+pIC at t=4- and 8-hours, respectively. (B) **p<006 and ***p<0.0001 for SP-R210_L(DN) vs SP-R210_L(DN)+IM at t=4- and 8-hours, respectively. (C) *p<0.015; **p<002, ***p<0.0002 for pIC treated SP-R210_L(DN) and SP-R210(KO) cells at t=4-8-hours. (C) *p<0.015; **p<002, ***p<0.0002 for pIC treated SP-R210_L(DN) and SP-R210(KO) vs untreated cells at t=4-8-hours. (D) *p<0.05; **p<0.006 for IM treated SP-R210_L(DN) and SP-R210(KO) vs untreated cells at t=4-8-hours.

Figure 3.. Differential cytokine kinetics in control and SP-R210-deficient cells.

Control, SP-R210_L(DN), and SP-R210(KO) cells cultured overnight in FBS replete DMEM were washed, placed in infection medium for 2-hours with no infection (NI) or with 4 MOI IAV PR8. Media were collected for the 2-hour time point and then replaced with 0.5 mL DMEM/10% FBS and media harvested at 2, 8, 10, and 24-hours. The concentration of IFNβ (A) and TNFα (B) was quantitated by ELISA. N=2 experiments performed in duplicate. All data are shown as means±SD. In (A) ⁺⁺⁺⁺, ****p<0.001 SP-R210_L(DN)-IAV vs SP-R210(KO)-IAV and Control cells-IAV and no infection controls at t=10-hours, ^##p<0.01 SP-R210_L(DN)-IAV vs SP-R210(KO)-IAV at t=24-hours, and ⁺⁺⁺⁺, ****p<0.001 ⁺⁺⁺⁺, ****p<0.001 SP-R210(KO)-IAV compared to Control cells-IAV and no infection controls at t=24-hours.

Figure 4.. Disruption of SP-R210_L but not both isoforms attenuates endosomal escape and expression of the viral genome.

(A and B) Selective depletion of SP-R210_L leads to sustained attenuation of IAV viral genome expression, whereas depletion of both isoforms results in temporary suppression of IAV genome expression. *p-value<0.05, **p-value<0.005, ***p-value<0.0005 and ****p-value<0.00005 for control vs. SP-R210_L(DN) cell comparisons; ^##p-value<0.005, ^####p-value<0.00005 for SP-R210(KO) vs. SP-R210_L(DN) cell comparisons. +p-value<0.05 for control vs SP-R210(KO) cell comparison. N=4 independent experiments performed in duplicate. Cells were infected with 4 MOI IAV PR8. Cells were stained with a biotinylated anti-NP antibody and NP stained cells visualized using a PE-Cy5 secondary antibody. (C) Lack of SP-R210_L limits expression and nuclear accumulation of NP but not uptake of the virus in SP-R210_L(DN) cells. Control and SP-R210_L(DN) cells were cultured on 12 mm glass coverslips placed in 12-well dishes overnight at a density of 150,000 cells per well. Cells were washed in DPBS and infected with 10 MOI IAV PR8 at 4°C for 1.5-hours and then either harvested for flow cytometry analysis of bound virus on non-permeabilized cells, or returned at 37°C and infection allowed to proceed for 1.5- or 4-hours. Coverslips were the washed, fixed permeabilized in blocking buffer, and stained with DAPI to visualize nuclei or TRITC-conjugated anti NP antibodies. Stained cells were washed, and coverslips mounted on slides, and visualized on a Nikon epi-fluorescent microscope. Cells were infected with 10 MOI IAV PR8. (D) Lack of SP-R210 isoforms does not affect binding of IAV PR8 to isolated cell membranes. Binding of IAV PR8 to isolated cell membranes from control, SP-R210_L(DN), SP-R210(KO) cells was by ELISA using anti-hemagglutinin antibodies as described recently [48]. N=2 independent experiments performed in duplicate. (E) Lack of SP-R210 isoforms does not deplete α2,3-sialic receptors for IAV PR8 hemagglutinin, whereas SP-R210_L depletion results in increased α2,6-sialic acid residues on SP-R210_L(DN) cells. MAA lectin and SNA lectin were used to stain control, SP-R210_L(DN), and SP-R210(KO) to determine α2,3- and α2,6- on the cell surface by flow cytometry, respectively. **p-value<0.005 comparing WT to SP-R210_L(DN) cells; ^##p-value<0.005 comparing SP-R210(KO) to SP-R210_L(DN) cells. N=3 independent experiments. (F,G) Cells were cultured on coverslips and infected with PR8 IAV for 15-, 30-, 60-, or 240-minutes with 10 MOI IAV PR8 as described above. Cells were then fixed and co-stained with NP and Rab5 antibodies (upper row), Rab7 (middle rows), or LAMP1 antibodies (bottom row), and counterstained with DAPI to visualized nuclei. Internalized IAV transitioned through early (Rab5+) and late endosomes (Rab7+) endosomes (white arrowheads) and then nuclear entry in control cells (F) compared to accumulation in perinuclear Rab7+ vesicles (white arrowheads) and spatial overlap with LAMP1+ vesicles in SP-R210_L(DN) cells over time (G). Anti-mouse FITC-conjugated and anti-rabbit TRITC-conjugated secondary antibodies were used to visualize staining with NP and endolysosomal marker antibodies Images were acquired using a LEICA confocal microscope using IMARIS software.

Figure 5.. Depletion of SP-R210_L or both isoforms alters vesicular trafficking of endosomal markers and IAV.

(A-B) Increased uptake of Dextran in acidified vesicles in SP-R210_L(DN) cells compared to control and SP-R210(KO) cells. Cells were incubated with 125 μL of 50 μg/mL FITC- or 30 μg/mL pHRodo-labeled Dextran and cells harvested at indicated time points in this and following experiments were analyzed by flow cytometry. *p-value<0.05, and***p-value<0.0005 for WT vs. SP-R210_L(DN) cell comparisons; (C-D) Depletion of both SP-R210 isoforms enhances endocytosis (C) but not transport of transferrin (D) to acidified endolysosomes. Cells were incubated with 125 μL of 50 μg/mL FITC-Transferrin or 125 μL of 30 μg/mL pHRodo-Transferrin in 1:1 DMEM and uptake of transferrin assessed by flow cytometry. ^##p-value<0.005 between SP-R210_L(DN) and SP-R210(KO) cells; ⁺⁺p-value<0.005, and ⁺⁺⁺p-value<0.0005 for WT vs. SP-R210(KO) cell comparisons. (E) SP-R210-deficient cells exhibit similar uptake rate of IAV PR8. Cells were incubated with 15 MOI of AlexaFluor488-PR8 at 37°C in with 1:1 DMEM/PBS and uptake of FITC-PR8 assessed by flow cytometry over time. N=3 independent experiments performed in duplicate. (F) Lack of SP-R210_L delays transport of IAV to acidified vesicles. Cells were incubated with 15 MOI of IAV PR8 at room temperature for 15-minutes, washed and incubated with 1:1 DMEM/PBS and at 37°C for the indicated times. ⁺p-value<0.05 and⁺⁺p-value<0.005 for WT vs.SP-R210(KO) cell comparisons. **p-value<0.005 between WT and SP-R210_L(DN) cells. N=4 independent experiments performed in duplicate (G-H) Depletion of SP-R210_L but not SP-R210_S leads to retention of IAV in acidified vesicles, whereas depletion of both isoforms appears to delay endosomal exit and replication of the viral genome at early stage of infection. Control, SP-R210_L(DN), and SP-R210(KO) cells were infected with 10 MOI IAV PR8 for 15-, 35-, 60-, 90- and 240-minutes. Cells were harvested, fixed, permeabilized, and stained with the pH-sensitive anti-hemagglutinin (HA) pHHA antibody clone (G),pH-insensitive HA antibody recognizing the HA trimer (H) or with secondary antibody only to subtract background signal . ^#p-value<0.05 for SP-R210_L(DN) and SP-R210(KO) cell comparisons; ⁺⁺⁺⁺p-value<0.00005 between WT and SP-R210(KO) cells and ****p-value<0.00005 for WT and SP-R210_L(DN) cell comparisons at each time point. N=8 independent experiments in experiments shown in G and n=2 and H, respectively. Data shown are means±SD. (I) Experimental design for experiments shown in G and H

Figure 6.. Macropinocytosis and macrophage activation in response to IAV infection are coupled in SP-R210_L(DN) but not control cells.

(A-F) Electron microscopic visualization revealed internalization of IAV in macropinocytic vesicles 1-hour after infection in control and SP-R210_L(DN) cells and in the process of engulfment by membrane ruffles (white arrows). IAV in the process of fusion in endosomes (A, open arrow) was seen in control cells and degradation in SP-R210_L(DN) cells (F, open arrow). En: endosome; Mp: macropinosome; MR: membrane ruffles; MPc: macropinosome cup. (G-O) Incubation with macropinocytic inhibitors of NHE1 (G; EIPA), PAK1 (H: IPA3.0), and RAC1 (I:EHT1864) attenuated inhibited IAV infection in both control and SP-R210_L(DN) cells in a concentration-dependent manner. The inactive PAK-1 analog PIR3.5 (J) and GEF Trio NSC23766 (K) had no effect. Inhibition of macropinocytosis by EIPA and EHT1864 blocked secretion of IFNβ in SP-R210_L(DN) cells (L, N) and TNFα in both cell lines (N,O). Inhibitors were added 30-minutes prior to infection with IAV PR8 and maintained for 2-hours during infection. Cells were then washed and infection allowed to proceed for 12-hours in DMEM/10%FBS. Media were harvested at the end of the incubation period for cytokine measurements by ELISA and cells harvested for flow cytometry analysis of infection. N=3 independent experiments in duplicate. *p<0.05, **p<0.005 for % NP+ cells and cytokine comparison for each cell line. ^$$p<0.005 for cytokine comparisons between cell lines at t=0. ^#p<0.05 and ^##p<0.005 for TNFα comparisons to t=0 in control cells.

Figure 7.. Differential RhoGTPases and guanine nucleotide exchange factors in SP-R210-deficient macrophages.

(A-C) Western blotting (A) and densitometric analysis of SP-R210 isoforms (B) and RAC1 (C) on isolated cell membranes from control, SP-R210_L(DN), and SP-R210(KO) macrophages. *p<0.01, ****p<0.0001. (D-I) Western blotting (D) and densitometric analysis of RAC2 (E), CDC42 (F), ARHGEF1 (G), VAV1 (H), and VAV3 (I). *p<0.01, *p<0.001. ***p<0.0001. ****p<0.00001, n=3 experiments performed in triplicate. All data are shown as means±SD.

Figure 8.. IFITM3 is increased and contributes to endosomal restriction of the IAV in SP-R210_L(DN) cells.

(A) Basal and IAV-induced expression of IFITM3 in SP-R210_L(DN) cells. Control and SP-R210_L(DN) cells were infected with PR8 IAV at 4 MOI, samples were collected at 3-, 6-, 12-, and 24-hours post infection, and processed for Western blotting and densitometry analysis of IFITM3 expression. GADPH was probed as loading control. (B) Control and SP-R210_L(DN) cells were transiently transfected with IFITM3 siRNA and knockdown confirmed by western blotting. (C) IFITM3 siRNA knockdown partially restores expression of IAV viral genomes in SP-R210_L(DN) cells, without effect on WT cells. *p-value<0.05 as indicated, n=3 experiments. (D) Knock down of IFITM2 had no effect on IAV infection. Infection of siRNA transfected cells was assessed 24-hours after addition of virus,

Figure 9.. Depletion of both SP-R210 isoforms enhances endosomal localization of IFITM3 during IAV infection.

Control, SP-R210_L(DN), and SP-R210(KO) cells cultured on glass coverslips were infected with 4 MOI of PR8 for 15- and 30-minutes. Cells were fixed, permeabilized, and stained for IFITM3 with either Rab5 (A) or Rab7 (D) to visualize early and late endosomes, respectively. Images were captured using a Nikon Confocal microscope. Colocalization analysis of fluorescent images of 20 random cells was performed using Pearson’s correlation coefficient (PCC) (B, E) or intensity correlation quotient (ICQ) (C,F). Significant differences were observed only for IFITM3/Rab7 colocalization at 15-minutes after infection in SP-R210(KO) cells compared to WT and SP-R210_L(DN) cells with transient declines in the latter (D,E,F). *p<0.05, ^$$p<0.01 at 15-minutes between SP-R210KO vs control and SP-R210_L(DN), respectively, based on 20 cells per cell line in random microscopic fields. Data are means±SD.

Figure 10.. Working model of SP-R210 isoform-mediated macropinocytosis of IAV.

Binding of IAV to cell membrane and SP-R210 isoforms elicits macropinocytic membrane projections and macropinocytic cup formation in a RAC-dependent manner leading to internalization of the virus in macropinosomes. SP-R210_L inhibits acquisition of TLR7+ endosomes during macropinosome acidification in an IFITM3-dependent manner inhibiting virus sensing in control cell macropinosomes or endosomes. Viral genome exits and then replication and transcription of the viral genome proceeds unimpeded in control cells. On the other hand, SP-R210_S triggers macrophage activation during macropinocytic entry and IFITM3-dependent containment of the virus in TLR7+ macropinosomes of increasing acidity where degradation of the virus facilitates virus sensing and amplification of the anti-viral response in macrophages. Image was generated using BioRender (www.BioRender.com).