Abstract
Pseudomonas aeruginosa is a significant Gram-negative opportunistic bacterium that causes a spectrum of organ system diseases, particularly in immunocompromised patients. This bacterium has been shown to induce unfolded protein response (UPR) during mammalian infection. Annexin A2 (AnxA2) is a multi-compartmental protein relating to a number of cellular processes; however, it remains unknown whether AnxA2 coordinates a UPR pathway under bacterial infection conditions. Here, we report that the endoplasmic reticulum (ER) stress IRE1-XBP1 pathway was upregulated by AnxA2 through p38 MAPK signaling following P. aeruginosa infection in macrophages, while ATF4 and ATF6 not. In addition, XBP1 was found as a positive regulator of innate immunity to tame P. aeruginosa challenges by enhancing autophagy and bacterial clearance. XBP1 also facilitated NF-κB activation to elicit the release of pro-inflammatory cytokines predominantly in macrophages. Together, our findings identify AnxA2 as a regulator for XBP1-mediated UPR pathway.
Keywords: Pseudomonas aeruginosa, ER stress, Annexin A2, XBP1
Summary:
AnxA2 plays a role in IRE1-XBP1 pathway of ER stress induced by P. aeruginosa infection through inhibition of phospho-p38 MAPK.
Graphical Abstract
Introduction
The endoplasmic reticulum (ER) plays a vital role in protein folding and transportation. In mammalian cells, 3 unique ER stress sensors, protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) are found functioning in unfolded protein response (UPR). ER stress contributes to PERK self-activation to phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) [1], which in turn increases the translation of ATF4. In addition, ATF4 targets C/EBP homologous protein (CHOP) involved in ER stress-mediated apoptosis [2]. Accumulation of misfolded proteins also contribute to the cleavage of ATF6 precursor, which will be transported into the nucleus to regulate the transcription of the underlying ER molecules [3–5]. IRE1 possesses an endonuclease and a trans-autophosphorylation of kinase domain. Upon activation by ER stress, IRE1 cleaves mRNA of X-box binding protein 1 (XBP1) and remove its 26 nucleotides (intron) [6, 7], then the sliced-XBP1 (XBP1-s) can be encoded successfully as a transcription factor to modulate the ER chaperones and ER-associated degradation (ERAD) genes to promote protein folding [8].
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium and causes a serious infection in immunocompromised patients, developing acute or chronic infection in the lung and other organs [9–13]. Due to increasing multidrug resistance, treatment of P. aeruginosa infection is extremely challenging. Previous studies indicated that P. aeruginosa was involved in ER stress in primary bronchial epithelial cells and Caenorhabditis elegans [14–17], but little is known whether and how P. aeruginosa modulates ER stress in mammalian macrophages. AnnexinA2 (AnxA2) is a member of calcium-dependent phospholipid-binding protein family, functioning in various biological processes, including autophagy, apoptosis, cell motility, and cancer development [18–22]. Our previous study indicated the role of AnxA2 in modulating P. aeruginosa infection [18]; however, it remains unknown whether and how AnxA2 cross-talks with UPR signaling under P. aeruginosa infection.
Here, we sought to elucidate the effect of AnxA2 on UPR function in P. aeruginosa - infected macrophages. Our results indicate that AnxA2 may regulate XBP1-mediated UPR pathway, which is independent of ATF4 and ATF6. Mechanistically, XBP1 promotes autophagy and NF-κB nuclear translocation to promote inflammatory cytokines in macrophages after bacterial infection.
Materials and Methods
Mice
Male and female C57BL/6J mice (6–8 weeks) were obtained from The Jackson Laboratory (Bar Harbor, ME). Anxa2−/− mice were kindly provided by Dr. K. Hajjar (Cornell University). Animals were kept in pathogen-free facility at University of North Dakota. All animal studies were approved by the University of North Dakota Institutional Animal Care and Use Committee IACUC) and performed in accordance with the NIH animal care and institutional guidelines.
Cells
MH-S alveolar macrophage cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA). A549 and MLE-12 were maintained in the lab. Primary alveolar macrophages (AMs) were isolated from bronchoalveolar lavage fluid (BALF). Briefly, mice were anesthetized with 45 mg/kg ketamine, sacrificed, and then made a small incision in the trachea. Lungs were washed 3 times with 1 ml PBS containing 1% FBS. The BALF was centrifuged at 600×g for 5 min at 4°C. Cell pellets were resuspended in RPMI 1640 medium with 10% FBS and incubated for 1 h in 37°C/5% CO2 incubator. Non-adherent cells were removed by washing with PBS.
Bacteria and infection
The P. aeruginosa wild type strain, PAO1, was kindly provided by Dr. S. Lory (Harvard University). PAO1-mecherry strain was stored in our lab. PAO1-EGFP was obtained from Dr. G. Pier (Harvard University). Escherichia coli DH5-α was obtained from New England Biolabs (Ipswich, MA). Bacteria were grown in lysogeny broth medium at 37°C for 16~18 h. Bacteria were then centrifuged at 5000×g for 10 min and resuspended with PBS. Mammalian cells were infected with bacteria at multiplicity of infection (MOI) of 20 for 2 h under antibiotic-free medium.
Quantitative real-time PCR
RNA was isolated from cell or tissue samples by TRIzol following the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, MA). qPCR assay was performed using SYBR Green qPCR Master Mix (ThermoFisher Scientific) accompanied with specific primers in the CFX Connect Real-Time PCR Detection System (Bio-Rad). Relative expression of mRNA was normalized with β-actin.
Transfection
AnxA2 and XBP1 small interfering RNAs (siRNAs) were obtained from ThermoFisher Scientific and siRNA p38 was obtained from Cell Signaling Technology. MH-S or AMs were transfected with 50 nM siRNA using RNAiMAX (ThermoFisher Scientific) for 24 h following the manufacturer’s instructions. pFLAG.XBP1u. CMV2 and pFLAG.XBP1p. CMV2 plasmids were obtained from Addgene. Plasmids were transfected using Lipofectamine 3000 (ThermoFisher Scientific) for 24 h following the manufacturer’s instructions.
Immunoblotting
Mouse mAbs against actin, Anxa2, ATF-6α, IRE1α, p50, p-p50, p65, p-p65, XBP-1, rabbit polyclonal Ab p-PERK (Thr 981), and goat polyclonal Ab PERK were obtained from Santa Cruz Biotechnology (Dallas, TX). Rabbit mAbs against Anxa2, ATF4, BiP, CHOP, IRE1α, p38 MAPK, p-p38 MAPK, and XBP-1s were obtained from Cell Signaling Technology. Rabbit polyclonal Ab against p-IRE1α (Ser 724) was obtained from Invitrogen (Carlsbad, CA). Rabbit polyclonal Abs against calnexin was obtained from Sigma-Aldrich (St. Louis, MO). Cell and lung samples were lysed in the RIPA with protease inhibitor. Protein concentration was quantitated with the BCA Protein Assay Kit (ThermoFisher Scientific). SDS Loading Buffer was added into the lysate, and then boiled for 10 min at 95°C. Proteins were separated by electrophoresis on 10 – 15% SDS-PAGE gels and transferred onto nitrocellulose membrane. Then membrane was blocked for 1 – 2 h at room temperature using 5% nonfat milk. Proteins were detected with first (4°C overnight) and second antibody (room temperature 2 h) at indicated concentrations. Protein bands were visualized using ECL reagent and quantified by Quantity One software (Bio-Rad).
Phagocytosis Assay
The cells were plated in 24-well plates overnight and challenged with GFP-PAO1 at 10:1 MOI for 1 h. Then, the cells were washed with PBS and treated with 100 μg/ml polymyxin B for 1 h to kill extracellular bacteria. Phagocytosis of bacteria was measured using a Synergy HT fluorometer (BioTek, VT) with 485 ± 20 nm excitation and 528 ± 20 nm emission filters.
Bacterial internalization and killing
Cells were infected with P. aeruginosa at a 1:20 MOI for 2 or 4 h. After 2 h of incubation at 37°C, the wells were washed and treated with 100 μg/mL polymyxin B for 1 h to kill extracellular bacteria. The cells were then washed in PBS and lysed. The bacterial CFUs were counted using LB agar dishes.
ELISA
Cytokine levels of TNF-α, IL-1β, and IL-6 were measured using ELISA kits following the manufacturer’s instructions.
Immunofluorescent assay
Cell samples were washed with PBS and fixed with 4% paraformaldehyde for 20 min at 4°C. Cells were permeabilized using 0.2% Triton X-100 for 20 min and blocked using 5% BSA for 20 min at room temperature. Cells were incubated with corresponding first antibody and second antibody (diluted by 5% BSA). Images were observed under an LSM 510 Meta confocal microscope.
Statistical analysis
Most experiments were conducted in triplicate and repeated at least three times. Data were presented as mean percentage and compared by one-way ANOVA (Tukey post hoc test) using GraphPad Prism 5 software.
Results
Pseudomonas aeruginosa modulates the UPR pathway in macrophages
To explore whether P. aeruginosa participates in ER stress process in macrophages, mouse alveolar macrophage cell line (MH-S) was infected with P. aeruginosa at different multiplicity of infections (MOIs) and different time points. Thapsigargin (Tg, 200 nM) is an inducer of ER stress by rapidly activating all 3 UPR pathways within 2 h [23]. Our immunoblotting data showed that P. aeruginosa infection was capable of inducing all UPR pathways in MH-S (Fig 1A–B and Fig S1A–B). Further immunoblotting analysis also confirmed similar patterns in THP-1 (Fig 1C and Fig S1C). We then observed that transcription of ATF4, GADD34, and XBP1-s was significantly increased in primary mouse alveolar macrophages (AMs) under P. aeruginosa infection, whereas other ER stress-related molecules, including BiP, CHOP, and ATF6, were not altered (Fig. 1D–I). Comparable results were also found in MH-S (Fig. S1D–I). Furthermore, we found that PAO1-GFP internalized in AMs and colocalized with calnexin (CANX), an ER-integrated protein modulating protein folding and assembly (Fig. S2B). Invasion by P. aeruginosa was also determined in both mouse and human epithelial cells (MLE-12 and A549) (Fig. S2A). Pretreatment with phagocytosis inhibitor Latrunculin A could also block the internalization of P. aeruginosa (Fig. S2C). Collectively, these data suggest that P. aeruginosa is involved in modulating ER stress.
Figure 1. P. aeruginosa induced ER stress in macrophages.
(A) MH-S was infected with P. aeruginosa at different multiplicity of infections (MOIs) (1, 5 10, and 20) or 50 μM Thapsigargin (Tg) for 2 h. Immunoblot analysis of UPR signaling pathways.
(B, C) MH-S and THP-1 were infected with 20 MOI P. aeruginosa for 30 min, 60 min, or 120 min or 50 μM Tg for 2 h. Immunoblot analysis of UPR signaling pathways.
(D-I) AMs were infected with 20 MOI P. aeruginosa for 30 min, 60 min, or 120 min or 50 μM Tg for 2 h. qPCR measuring the mRNA levels of (D) BiP, (E) ATF4, (F) CHOP, (G) GADD3, (H) ATF6, and (I) XBP1-s.
(A-C) was repeated at least twice. qPCR data of (D-I) are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
AnxA2 loss impairs P. aeruginosa-induced ER stress in macrophages
To elucidate whether AnxA2 participates in P. aeruginosa-induced ER stress, AnxA2 was silenced using siRNA technology (Fig. 2A). Immunoblotting results showed that XBP1-s was significantly reduced in AnxA2-silenced MH-S and THP-1 cells (Fig. 2B and Fig. S3A), while no difference was found surrounding ATF4 and ATF6 pathways (Fig. 2B). Additional immunofluorescence analysis revealed that AnxA2 was localized around the ER and colocalized with CANX (Fig. S3B). Furthermore, qPCR analysis confirmed that transcription of BiP was reduced slightly in AnxA2-silenced MH-S following P. aeruginosa infection (Fig. 2C), but ATF4, CHOP, and ATF6 levels not (Fig. 2D–F). Moreover, transcription of XBP1-s was significantly reduced in AnxA2-silenced MH-S (Fig. 2G). Similarly, the level of XBP1-s was also down-regulated in AnxA2 KO mice lung tissue compared to wild type lung tissue and AnxA2-silenced A549 cells challenged with P. aeruginosa for 48 h (Fig. 2H and Fig. S3C). Consistent with our previous study, AnxA2 KO mice showed higher lethality compared to wild type mice in response to P. aeruginosa infection (Fig. 2I) [18]. Together, these data indicate that AnxA2 may be a potential regulator of ER stress-associated IRE1-XBP1 pathway.
Figure 2. AnxA2 promoted P. aeruginosa-induced UPR in IRE1-XBP1 pathway in macrophages.
MH-S cells were transfected with 50 nM control siRNA and AnxA2 siRNA for 24 h, then treated with or without 20 MOI P. aeruginosa or 50 μM Tg for 2 h. (A-B) Immunoblotting analysis of (A) AnxA2, (B) BiP, ATF4, ATF6, XBP1-s, and β-actin. (C-G) MH-S cells were treated as (A). qPCR measuring the mRNA levels of BiP, ATF4, CHOP, ATF6, and XBP1-s.
(H) Immunoblotting analysis of ATF4, ATF6, XBP1-s and β-actin in P. aeruginosa infected WT and Anxa2−/− mouse lung tissues.
(I) WT and Anxa2−/− mice were challenged with 1×107 CFU P. aeruginosa and then observed up to 120 h. Kaplan–Meier survival curves were obtained. p=0.0081.
(A, B and H) was repeated at least twice. qPCR data of (C-G) are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
AnxA2 regulates XBP1-s through the p38 MAPK pathway
To further investigate whether AnxA2 regulates the IRE1-XBP1 pathway through p38 MAPK signaling, we measured the levels of p38 and phospho-p38. As shown in Fig. 3A, AnxA2 silencing enhanced the phosphorylation of p38. Further analysis showed that p38 silencing contributed to the phosphorylation of IRE1 and maturation of XBP1 (Fig. 3B). qPCR assays also demonstrated similar findings with mRNA transcripts (Fig. 3C). AMs were then pretreated with p38 MAPK inhibitor, SB202190, which is able to inhibit the activity of p38 MAPK by competing with ATP [24], and we found that SB202190 also increased the expression of XBP1-s, similar to the data through p38 silencing (Fig. 3C and D). Further immunoblotting analysis showed that production of IL-1β and IL-6 was also impaired by SB202190 under P. aeruginosa infection (Fig. S4A). Hence, these results indicate that AnxA2 mediates IRE1-XBP1 inhibition through phosphorylation of p38.
Figure 3. AnxA2 regulated XBP1-s through p38 MAPK pathway in macrophages.
(A) MH-S cells were treated as described in Figure 2A. Immunoblotting analysis of p-p38, p38, and β-actin.
(B) MH-S cells were transfected with 50 nM control siRNA and p38 siRNA for 24 h, respectively, and then treated with or without 20 MOI P. aeruginosa or 50 μM Tg for 2 h. Immunoblotting analysis of p-p38, p38, p-IRE1, IRE1, XBP1-s and β-actin.
(C) MH-S cells were treated as (B), qPCR measuring the mRNA levels of XBP1-s.
(D) MH-S cells were pretreated with DMSO or 20 μM SB202190 (p38 inhibitor), and then treated with or without 20 MOI P. aeruginosa or 50 μM Tg for 2 h. qPCR measuring the mRNA levels of XBP1-s.
(A, B) was repeated at least twice. qPCR data of (C-D) are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
XBP1 participates in inflammatory response
Prior studies revealed that AnxA2 facilitates the production of proinflammatory cytokines [18]. Our data also affirmed the role of AnxA2 in promoting the production of proinflammatory cytokines (Fig. S4B–D). As XBP1 is downstream of AnxA2, we next investigated the function of XBP1 in inflammatory responses in macrophages. We noticed that XBP1-silenced MH-S cells exhibited reduced transcription of TNFα, IL-1β and IL-6 (Fig. 4A–C). Also, ELISA analysis verified that the production of TNFα, IL-1β and IL-6 was significantly inhibited in XBP1-silenced MH-S cells (Fig. 4D–F). In addition, we found that overexpressed XBP1 in MH-S stimulated the transcription of TNFα, IL-1β and IL-6 under P. aeruginosa infection (Fig. 4G, H). Together, these data reveal the opposite role of AnxA2 and XBP1 in inflammatory response.
Figure 4. XBP1 participated in inflammatory response.
(A-C) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h with or without P. aeruginosa infection for 2 h (mock infection-PBS as control). qPCR measuring the mRNA levels of TNFα, IL-1β, and IL-6.
(D-F) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h with or without P. aeruginosa infection for 2 h. qPCR measuring the mRNA levels of TNFα, IL-1β, and IL-6. ELISA measured the production of TNFα, IL-1β and IL-6.
(H-G) MH-S cells were transfected with empty plasmids, p-XBP1-U or p-XBP1-S for 24 h with P. aeruginosa infection for 2 h (PAO1 infection alone group without plasmid transfection as controls). qPCR measuring the mRNA levels of TNFα, IL-1β and IL-6. qPCR and ELISA data are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
XBP1 promotes the activation of NF-κB signaling
Extensive studies showed that NF-κB is a transcription regulator of proinflammatory cytokine genes. To explore whether XBP1 regulates the activation of NF-κB, we used siRNA to knockdown the expression of XBP1 in MH-S cells. qPCR and immunoblotting analyses indicated that the transcription and phosphorylation of the NF-κB p50 or p65 subunit were significantly reduced in the XBP1-silenced group, respectively (Fig. 5A–C, Fig. S4E). To further validate these results, we next detected the effect of XBP1 on NF-κB p-p65 nuclear translocation. Imaging analysis demonstrated that siXBP1 virtually abolished the translocation of NF-κB into the nucleus (Fig. 5D). Moreover, overexpression of XBP1 not only enhanced the expression and phosphorylation of NF-κB but also promoted its nuclear translocation (Fig. 5E–H). In summary, our observations suggest that XBP1 exerted a positive effect on enhancing the transcription of proinflammatory cytokines through activation of NF-κB.
Figure 5. XBP1 promoted activation of NF-κB.
(A) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h and then treated with or without 20 MOI P. aeruginosa for 2 h. Immunoblotting analysis of p50, p-p50, p65 and p-p65.
(B, C) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h and then treated with or without 20 MOI P. aeruginosa for 2 h. qPCR measuring the mRNA levels of p50 and p65.
(D) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h and then treated with or without 20 MOI P. aeruginosa for 2 h. Cells were stained with p-p65 antibody, followed by corresponding fluorescent antibody, p-p65 (green), DAPI (blue). Scale bars, 5 μm.
(E) MH-S cells were transfected with empty plasmids, p-XBP1-U or p-XBP1-S for 24 h or 48h. Immunoblotting analysis of p50, p-p50, p65 and p-p65.
(F, G) MH-S cells were transfected with empty plasmids, p-XBP1-U or p-XBP1-S for 24 h or 48h. qPCR measuring the mRNA levels of p50 and p65.
(H) MH-S cells were transfected with empty plasmids, p-XBP1-U or p-XBP1-S for 24 h; p-p65 translocation was visualized using immunostaining, p-p65 (green), DAPI (blue), Scale bars, 5 μm.
(A, E) was repeated at least twice. qPCR data of (B, C, F, G) are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
XBP1 is involved in the immunocompetence of macrophages
To further characterize the role of XBP1 in host defense against bacteria, we elucidated the effect of XBP1 on phagocytosis or bacterial killing in MH-S cells. We found that XBP1 silencing suppressed bacterial killing in MH-S cells (Fig. 6A–B), while XBP1 had no influence on phagocytic potency (Fig. 6C–D). Critically, overexpression of XBP1 also enhanced bacterial killing potency in macrophages (Fig. 6E, F). Further assessment indicated that XBP1 deficiency blocked the activation of autophagy induced by P. aeruginosa infection (Fig. S4F). Collectively, these data suggest that XBP1 acts as a positive regulator of innate immunity in restricting P. aeruginosa infection by autophagy.
Figure 6. XBP1 was involved in the immunocompetence of macrophage.
(A-B) MH-S cells were transfected with 50 nM control siRNA and XBP1 siRNA for 24 h with P. aeruginosa infection for 2 h or 4h. Intracellular P. aeruginosa clearance was determined.
(C-D) Phagocytosis of P. aeruginosa was determined at 2 h by colony forming unit (CFU) assay after PAO1-GFP challenge at MOI 10:1 for 2 h in XBP1-silenced or overexpressed MH-S cells.
(E-F) MH-S cells were transfected with empty plasmids, p-XBP1-U or p-XBP1-S for 24 h. Intracellular P. aeruginosa clearance was determined by CFU assay after P. aeruginosa challenge at MOI 10:1 for 2 h or 4 h.
Data are representative of three independent experiments (one-way ANOVA with Tukey post hoc test; *p < 0.05, **p < 0.01, *** p < 0.001).
Discussion
P. aeruginosa is considered as a significant gram-negative extracellular pathogen [25]. However, P. aeruginosa may be able to enter into nonphagocytic cells at low rates, which relies on the interaction between T6SS effector VgrG2b and γTuRC [26–28]. Furthermore, macrophages are professional phagocytes chasing and engulfing the invading bacterium [29]. Here, we present evidence supporting the internalization of P. aeruginosa. We also noticed that P. aeruginosa was colocalized with ER, indicating that the internalization of P. aeruginosa may directly disrupt ER. However, it remains to be further investigated how P. aeruginosa interacts with UPR.
ER stress is important for immune response to bacterial infection [15, 30]. AnxA2 has been reported to be involved in multiple cellular processes, including autophagy, growth and carcinogenesis [21, 22]. AnxA2 was found existing in the extracellular space. Previous reports indicated that AnxA2 may not only medicate the internalization of P. aeruginosa but also bind TLR4 mediating the downstream signal pathway [31, 32]. However, it is unknown whether AnxA2 participates in UPR modulation. Here, we observed that P. aeruginosa infection induced the IRE1-XBP1 pathway. Additional AnxA2 silencing revealed that AnxA2 enhanced XBP1 maturation following P. aeruginosa infection, hinting that AnxA2 may regulate protein folding process.
It has been reported that activation of IRE1-XBP1 is dependent on p38 MAPK in worms and in primary bronchial epithelial cells upon P. aeruginosa infection [14, 15]. However, the role of p38 MAPK in UPR pathway in mammalian macrophages is essentially unknown. We noticed that AnxA2 silencing led to increased phosphorylation of p38 after P. aeruginosa infection, consistent with previous reports [31, 33]. Additionally, p38 siRNA and p38 inhibitor promoted the activation of phosphorylation of IRE1 and activation of XBP1 in macrophages. Our qPCR data also revealed that p38 suppression significantly promoted the IRE1-XBP1 pathway in macrophages, indicating that AnxA2 is involved in IRE1-XBP1 pathway through p38. It is well known that Tg modulates UPR by disrupting ER Ca2+ homeostasis and leading to ER Ca2+ depletion. AnxA2 is reported containing a Ca2+ binding site and is activated in a Ca2+ dependent manner [34]. Our results confirmed that activated IRE1-XBP1 pathway by Tg is inhibited by AnxA2 deficiency, indicating the important role of AnxA2 in sensing Tg. Additionally, our data showed that p38 altered UPR but in a different fashion in macrophages compared to other models, such as worms and epithelial cells [31, 33]. This may be due to different cell types and perhaps the different experimental conditions. Epithelial cells are important and emerging immune cells [35, 36], but ultimately it is the macrophage that performs the phagocytosis and secretes high levels of inflammatory cytokines to launch fierce immune defense against both viral and bacterial infection [37, 38].
XBP1 is one of the important transcription factors that modulate a series of genes expression during innate immune response [30]. Additionally, we noticed that AnxA2 suppressed production of inflammatory cytokines in macrophages upon P. aeruginosa infection, indicating that although AnxA2 mediated activation of IRE1-XBP1 pathway, AnxA2 may modulate cytokine production independent of XBP1. Our previous work showed a suppressive role of AnxA2 in proinflammatory process in anxa2−/− mouse models. AnxA2 deficiency exhibited enhanced expression and translocation of NF-κB via TLR4-TRAM-TRIF pathway [18, 31]. Further study showed that ROS production and IL-17 signaling are also involved in AnxA2-mediated inflammatory signaling [39]. It is known that both AnxA2 and XBP1 are necessary for host defense against P. aeruginosa infection [15, 18]. Since the opposite roles of IRE1-XBP1 and AnxA2 in inflammation, AnxA2 may alter the outcomes of P. aeruginosa infection independent of XBP-1-mediated inflammatory pathway. Thus far, accumulating studies support the role of AnxA2 in autophagy activation and AnxA2 is important in autophagy-mediated bacterial clearance after P. aeruginosa infection [18, 40]. In this study, we also revealed that XBP1, mediating the activation of autophagy [41, 42], is essential for killing phagocytized P. aeruginosa. Together, those data suggest that AnxA2 may mediate the degradation of P. aeruginosa through XBP1-mediated autophagy mechanisms.
In conclusion, our findings characterized that AnxA2 mediates IRE1-XBP1 pathway through p38 MAPK during bacterial infection. Limitations of the study also exist. Although AnxA2 exhibited positive roles in IRE1-XBP1 pathway, the detailed mechanisms remain elusive. Is AnxA2 participating IRE1-XBP1 pathway through Ca2+? Is AnxA2 involved in protein synthesis? The detailed roles of AnxA2 in p38 and p38 in XBP1 are also unclear. It is known that C-terminal XBP1 SUMOylation enhances its transcriptional activity towards UPR target genes [43]. Further study is necessary to explore the underlying modulation mechanism of XBP1 SUMOylation under P. aeruginosa infection. Additionally, what is the roles of XBP1 in a KO mouse model or conditional KO mouse model against P. aeruginosa or other extracellular bacterial infection?
Supplementary Material
Acknowledgements
We thank Sarah Abrahamson (University of North Dakota Imaging Core Facility) for help with imaging. We also thank Dr. K. Hajjar for kindly providing Anxa2−/− mice.
Funding:
This work was supported by National Institutes of Health 5R01AI109317–04, 1R01AI138203–01 to M.W., and the UND Imaging and Flow Cytometry Cores were supported by P20GM103442 and P20GM113123. We thank the Imaging Core of University North Dakota, especially Sarah Abramhanson for her technical assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Footnotes
Potential conflicts of interest
All authors: No reported conflicts.
Supplemental information
Supplementary Information includes 4 figures.
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