Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 2023 Nov 1;91(12):e00311-23. doi: 10.1128/iai.00311-23

Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli

Shanjana Awasthi 1,✉, Bhupinder Singh 1, Vijay Ramani 1, Nachiket M Godbole 1, Catherine King 1
Editor: Kimberly A Kline2
PMCID: PMC10714950  PMID: 37909750

ABSTRACT

Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against Escherichia coli. Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with E. coli and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of E. coli in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with E. coli in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.

KEYWORDS: surfactant protein A-derived peptide, Toll-like receptor 4, host defense, bacterial phagocytosis, inflammation, Escherichia coli

INTRODUCTION

Gram-negative bacterial lung infections are one of the leading causes of deaths worldwide. Acquisition of new virulence traits and antibiotic resistance by bacterial pathogens have contributed to an increase in the morbidity and mortality in recent years (1). Limited or no therapeutic options exist for management of the uncontrollable infections and associated complications. New therapeutic approaches are urgently needed for improving the clinical management of critically ill patients suffering from difficult-to-treat bacterial infections (2–4). An understanding of the host-pathogen interactions could facilitate identification of therapeutically viable targets and development of novel therapies for enhancing the protective mechanisms against infectious organisms. Previous research in our laboratory has demonstrated that the interaction between lung surfactant protein-A (SP-A) and Toll-like receptor 4 (TLR4) stimulates phagocytic uptake and simultaneously reduces inflammatory response against Gram-negative bacteria (5). However, it has not been possible to use full-length or native SP-A for therapeutic purposes. Thus, we delineated SPA4 peptide from C-terminal TLR4-interacting region of SP-A. The SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) interacts with activated TLR4 complex in the diseased state and enhances host defense eventually leading to reduced bacterial burden, inflammation, and injuries in a mouse model against Pseudomonas aeruginosa (6). Also, the SPA4 peptide treatment reduces inflammation and tissue injury against non-infectious Escherichia coli O111:B4- and P. aeruginosa 10-derived lipopolysaccharide (LPS) (7–9). Therapeutically administered SPA4 peptide exhibits pro-phagocytic and anti-inflammatory activity through its binding to activated TLR4 complex against bacterial and LPS stimuli. However, the cellular and molecular mechanisms of SPA4 peptide activity are not completely known. The first step in phagocytosis is the recognition of Gram-negative bacteria and internalization of TLR4 with bacteria. Thus, we studied the effect of the SPA4 peptide on subcellular distribution and co-localization of TLR4 with bacteria in a time-dependent manner. Upon phagocytic uptake, the lysis, clearance, and processing of bacterial antigens and TLR4 take place in phagolysosomes and other subcellular organelles such as endoplasmic reticulum (ER), Golgi body, and vesicles through yet unknown transport and retro-transport and molecular mechanisms (10, 11).

The ER has only recently been identified to play a role in phagocytosis by macrophages (12). Our recently published data demonstrated reduced gene expression of ER membrane protein complex 1 (EMC1) by 15-folds but increased gene expression of histone cluster 2 protein or Hist2h2aa2 or histone H2A by 6.5-folds in LPS-challenged dendritic cells (7). Interestingly, the SPA4 peptide treatment reverses these changes in gene expression of EMC1 (increase by 10–20-folds) and histone H2A (decrease by 5–62-folds) against E. coli-derived LPS stimuli (7). The EMC1 is part of a multimeric ER membrane complex that engages with calnexin, a chaperone protein (13, 14). Both ER-specific calnexin and EMC1 proteins have been shown to orchestrate the phagosome formation and phagocytic uptake of a fungal pathogen (12, 14). Thus, for the first time, we determined the effect of SPA4 peptide treatment on expression and co-localization of calnexin and EMC1 with phagolysosome marker LAMP1 against E. coli.

It is established that while TLR4 recognizes its ligand or pathogen, it also stimulates the activity of NF-κB and AP-1 transcription factors, secretion of cytokine and chemokines, inflammasome, and other intracellular molecular partners. An exaggerated inflammatory response not only causes tissue damage and injury but also promotes proliferation of pathogens in the tissue microenvironment of injury. During inflammation, the activated AP-1 remodels chromatin for NF-kB-induced inflammatory response (15). The histone H2A, one of the four nucleosome proteins, is involved in the transcriptional regulation of host defense mechanisms. The SPA4 peptide reduces the P. aeruginosa-induced AP-1 and NF-κB, and E. coli-LPS-induced gene expression of histone H2A (6, 7). Based on these insights, we studied the cellular histone H2A protein expression and secreted levels of TNF-α in E. coli-challenged and SPA4 peptide-treated cells.

Our results demonstrate no binding or direct killing of E. coli by SPA4 peptide treatment. Instead, it increases bacterial uptake and lysis but suppresses TNF-α release in dendritic cell system against E. coli. Furthermore, TLR4 overexpression augments SPA4 peptide activity against bacterial stimuli. These results substantiate the immunomodulatory activity of SPA4 peptide against Gram-negative bacterial pathogens (E. coli and P. aeruginosa PA01 [previously published results]) expressing LPS and other distinct virulence characteristics (6). An increased phagolysosome formation and intracellular co-localization of TLR4 with E. coli inside the phagolysosome at early time point substantiate the TLR4-dependent pro-phagocytic activity of the SPA4 peptide. A slightly increased expression of calnexin and an increased co-localization of calnexin and EMC1 with phagolysosome marker LAMP1 in SPA4 peptide-treated cells indicate the role of ER in enhanced bacterial uptake and clearance. Simultaneously, the expression of histone H2A and secreted amounts of TNF-α are suppressed after treatment with SPA4 peptide. Together, the results presented here provide initial insights into the potential interplay of ER and histone proteins in TLR4-modulatory activity of the SPA4 peptide against infectious stimuli.

MATERIALS AND METHODS

SPA4 peptide

The SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) was synthesized at Genscript, Piscataway, NJ. Fluorescein isothiocyanate (FITC) was conjugated at the N-terminal end of the SPA4 peptide (FITC-SPA4; Genscript, Piscataway, NJ). The purity of each batch of peptide was confirmed by mass spectroscopy and high-performance liquid chromatography (HPLC). The SPA4 peptide was reconstituted in endotoxin-free water at the concentration of 834.2 µM and stored at −20°C.

Bacterial strains

The Escherichia coli 19138 serotype O6:K2:H1 (ATCC, Manassas, VA, USA) strain previously utilized to induce lung infection in mice (16) was included. The bacterial cultures were maintained in Luria-Bertani broth or agar media. Heat-killed E. coli 19138 was included in some of the experiments for which the bacterial suspension in mid-logarithmic growth phase was heated at 100°C for 30 min. E. coli 19138 was also transformed with pGFPuv plasmid DNA (Clontech Lab, Mountain View, CA) encoding green fluorescent protein (GFP) (17). The GFP expression was visualized using ultraviolet light. The biochemical characteristics of E. coli were determined using oxidase and catalase reagents (BD Biosciences, San Jose, CA) per the manufacturer’s instructions. The Gram staining was performed to ensure the purity of the bacterial cultures. The bacterial stock cultures were maintained in 15% glycerol at −80°C.

The pHrodo-labeled E. coli K-12 bioparticles (Thermo Fisher Scientific, Waltham, MA) were also included. The phagocytic uptake of pHrodo-labeled E. coli was determined by red fluorescence of bacteria due to their internalization in acidic phagolysosomes, as described previously (5, 6, 18).

Bacterial growth curve

Bacteria were grown in culture medium overnight at 37°C with shaking. The overnight bacterial cultures were subcultured in pre-warmed fresh medium and further incubated at 37°C. An aliquot of culture was removed at different time intervals, and its optical density was read at 600 nm (OD600). At the same time, the culture was serially diluted in sterile Dulbecco’s phosphate buffered saline (DPBS; Life Technologies, Grand Island, NY) and plated onto agar plates. Bacterial colonies were counted, and a linear regression equation was determined by plotting CFU/mL versus OD600. For subsequent experiments, the bacterial cultures were collected from the mid-logarithmic growth phase.

Binding of SPA4 peptide to bacteria

We also examined whether the SPA4 peptide directly binds to live bacteria and affects the bacterial growth outside the cells (19). Bacteria harvested from mid-logarithmic growth phase were washed in endotoxin-free phagocytosis assay buffer containing 1 mM CaCl2, 1 mM MgCl2, and 1 mM HEPES and incubated with 10, 50, 75, and 100 µM of FITC-SPA4 peptide. The reaction mix was incubated at 37°C on a shaking water bath (85 rpm) for 45 min and swirled after every 15 min. Bacterial cells were washed three times with DPBS to remove free FITC-SPA4 peptide and then run on an Accuri flow cytometer (BD Biosciences, San Jose, CA). Polymyxin B is a cyclic cationic peptide antibiotic that binds to anionic lipids, specifically the Gram-negative bacterial LPS (20). The Oregon green 514-conjugated polymyxin B (Life Technologies, Grand Island, NY) was included as a positive control.

The binding of the FITC-SPA4 peptide or Oregon green 514-conjugated polymyxin B to bacterial cells was studied by confocal microscopy (6). All images were acquired with 63× oil immersion objective in a Zeiss confocal microscope and processed using the Zeiss ZEN 2011 program. Bacteria alone served as a negative control.

Direct anti-bacterial effects of SPA4 peptide

We assessed direct anti-bacterial activity of SPA4 peptide as described previously (6). An aliquot of mid-logarithmic culture of E. coli was pelleted at 5,000 × g at 4°C and washed with sterile DPBS. Diluted bacterial suspension was added to the wells of a Honeycomb 2 plate (Oy Growth Curves Ab Ltd., Helsinki, Finland) that contained 1, 10, or 100 µg/mL SPA4 peptide or an equivalent amount of vehicle. Treatment with ampicillin was included as positive control. Absorbance readings (OD600) were taken at 37°C every 15 min overnight using the Bio Screen C (Oy Growth Curves Ab Ltd., Helsinki, Finland). An aliquot of the culture was collected, serially diluted in DPBS, plated on agar plates, and incubated overnight at 37°C. Bacterial colonies were counted to obtain CFU/mL.

Cell culture and maintenance

The JAWS II dendritic cells (ATCC, Manassas, VA) were maintained in alpha-modified minimum essential medium (α-MEM; Cellgro, Manassas, VA) supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin (Life Technologies, Grand Island, NY), and 5 ng/mL of recombinant murine granulocyte macrophage colony-stimulating factor (Peprotech, Rocky Hill, NJ) (6).

Transient transfection of dendritic cells with plasmid DNAs encoding wild type or dominant negative forms of TLR4

The JAWS II dendritic cells were transfected with pDisplay vector control or the plasmid constructs encoding wild-type mouse TLR4 or dominant negative form of mouse TLR4 on the same vector backbone, using the TransIT-TKO transfection reagent (Mirus, Madison, WI) as described previously (6, 21). Changes in NF-κB reporter activity and secretion of TNF-α against highly purified E. coli O111:B5 LPS were determined for the overexpression or suppression of TLR4 in transfected cells (6, 21). Transiently transfected cells were washed and re-seeded at the density of 105 cells per well in Opti-MEM-reduced serum medium (Life Technologies, Grand Island, NY) for an assessment of localization of pHrodo-labeled E.coli inside the phagolysosomes and secretion of TNF-α.

Phagocytosis assays

We employed the following approaches to assess the phagocytic uptake of E. coli:

Cellular uptake of GFP-expressing E. coli

Dendritic cells were suspended in endotoxin-free phagocytosis assay buffer containing 1 mM CaCl2, 1 mM MgCl2, and 1 mM HEPES. One million dendritic cells were incubated with GFP-E. coli 19138 (at 1 cell: 100 bacteria, multiplicity of infection or MOI) with or without 1% normal mouse serum and 75 µM SPA4 peptide. Serum was included to discern pro-phagocytic activity of SPA4 peptide from complement and other factors. The reaction mix was incubated at 37°C for 45 min while shaking and swirling after every 15 min. Cells were finally washed three times with DPBS to remove free bacteria and run on an Accuri flow cytometer (BD Biosciences, San Jose, CA). Vehicle-treated cells and bacteria alone served as controls. The flow cytometric pattern of cells and bacteria was used for setting the gates. Any shift in the FL1 (green) fluorescence of the gated cells was considered for bacterial phagocytosis. The cells then were fixed and stained with Hoechst 33342 dye for nuclear staining. All the images were acquired using a 63× oil immersion objective in a Zeiss confocal microscope and processed using the Zeiss Zen program per our previously published method (6).

Localization of pHrodo-labeled E. coli in acidic phagolysosomes

The non-transfected or transiently transfected JAWS-II dendritic cells were seeded in Opti-MEM medium at a density of 1 × 105 cells per well. Sonicated pHrodo-conjugated E. coli (Thermo Fisher Scientific, Waltham, MA) were added to the cells (MOI ~1 cell: 2.0 ng bacteria or 1 cell: 10 bacteria). After 1.5 h of incubation, the cells were treated with 75 µM SPA4 peptide. Fluorescence readings then were taken after 3.5 h of incubation at 530 nm excitation and 590 nm emission wavelengths, using the Synergy 2 multi-mode microplate reader (Biotek, Winooski, VT). The percent localization of pHrodo-conjugated bacteria into acidic phagolysosomes was calculated per our published reports (5, 6). The cell-free supernatants were collected after taking the fluorometric readings and stored at −80°C for further analysis. The non-adherent and adherent cells were harvested, washed, homogenized, and subjected to measurement of cellular protein content using the Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, Waltham, MA).

Cellular expression, subcellular distribution, and co-localization of pHrodo-labeled E. coli and TLR4

After the phagocytosis assay for a total duration of 2.5–4.5 h, the cells were fixed in 4% paraformaldehyde solution for 10 min at room temperature. Fixed cells were washed thrice with a buffer containing 1% FBS and 0.05% saponin in DPBS and incubated with permeabilization buffer containing 10% FBS, 0.05% saponin, and 10 mM HEPES in α-MEM medium on ice for 20 min. The cells were then incubated with a blocking buffer containing 1% bovine serum albumin (BSA) at room temperature for 1 h followed by staining with 1:50 diluted rabbit anti-TLR4 antibody (10 µg/mL final concentration, Abcam, Waltham, MA) in 1% BSA overnight at 4°C and 1:200 diluted Alexa Fluor-488-conjugated secondary antibody (10 µg/mL final concentration, Abcam, Waltham, MA) at room temperature for 1 h. Finally, the cells were washed and stained with 1:1,000 diluted nuclear stain 2-[4-(aminoiminomethyl) phenyl]-1H-indole-6-carboximidamide hydrochloride (DAPI, 1 µg/mL final concentration, Thermo Fisher Scientific, Waltham, MA) at room temperature for 10 min. The chambered slide with stained cells was mounted with antifade mountant and incubated at room temperature overnight in the dark. The immunofluorescence staining was examined by Leica TCS SP8 confocal microscope. The images were collected with a 63× objective and analyzed with LASX software (Leica Microsystems Inc., Deerfield, IL). All intact cells with stained nuclei were annotated, and intracellular and cell-surface expression of TLR4 were noted at the mid-nuclear plane. Next, we determined the subcellular distribution of TLR4 (identified as green fluorescence) and its co-localization (as yellow fluorescence) with red fluorescent bacteria.

Expression and co-localization of ER proteins: calnexin and EMC1 with phagolysosome marker- LAMP1

The JAWS II cells were seeded at the density of 1 × 105 cells per well in an 8-well chamber slide and incubated for 30 min prior to challenge with heat-killed E. coli 19138 at the MOI of 10 (1 cell: 10 bacteria). After 1 h of infectious challenge, the cells were treated with 75 µM SPA4 peptide. The cells were incubated for a total period of 2.5 h at 37°C in 5% CO2 atmosphere, fixed in 4% paraformaldehyde solution, and immunostained overnight at 4°C in combination with 1:200 diluted antibodies for calnexin (2.5 µg/mL final concentration, Abcam, Waltham, MA) and LAMP-1 (2.5 µg/mL final concentration, Thermo Fisher Scientific, Waltham, MA), respectively. The cells were washed and immunostained with 1:200 diluted anti-rabbit Alexa Fluor-488-conjugated antibody (10 µg/mL final concentration, Abcam, Waltham, MA) and 1:200 diluted anti-mouse Alexa Fluor-568-conjugated antibody (10 µg/mL final concentration, Abcam, Waltham, MA). The cells then were stained with the nuclear stain DAPI and analyzed by confocal microscopy.

In a separate set of experiments, the cells were immunostained with a combination of 1:100 diluted polyclonal antibodies raised in rabbit against 885–965 amino acids of human EMC1 (10 µg/mL final concentration, Abclonal, Woburn, MA) and LAMP-1 (5 µg/mL final concentration, Thermo Fisher Scientific, Waltham, MA). The expression and co-localization of calnexin, EMC1, and LAMP1 were studied in E. coli-challenged or -unchallenged, SPA4 peptide- or vehicle-treated cells in comparison with unstained cells or cells stained with DAPI (nuclear stain) alone.

Expression of calnexin, EMC1, histone H2A, and LAMP1 by immunoblotting

The JAWS II cells were seeded at the density of 1 × 105 cells per well in a 24-well plate and incubated for 30 min prior to challenge with heat-killed E. coli 19138 at the MOI of 10. After 1 h of infectious challenge, the cells were treated with 75 µM SPA4 peptide or an equivalent volume of vehicle. The cell lysates were prepared in a homogenization buffer (1× RIPA lysis buffer containing 0.5 µg/mL leupeptin, 0.68 µg/mL pepstatin, 0.2 mM phenylmethylsulfonyl fluoride, 0.01 mM sodium fluoride, and 0.2 mM sodium orthovanadate) at 2.5 h of seeding the cells. Total cellular protein (2–5 µg) was loaded per well and separated by SDS-PAGE on 4%–20% gradient gel (Thermo Fisher Scientific, Waltham, MA). Separated proteins were transferred onto nitrocellulose membrane, and non-specific sites were blocked with Blotto buffer (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. The membrane with transferred proteins was immunoblotted with 1:500 or 1,000 diluted primary antibodies for calnexin (Abcam, Waltham, MA), EMC1 (Abclonal Technology, Woburn, MA), histone H2A (Cell Signaling Technology, Danvers, MA), and LAMP1 (Thermo Fisher Scientific, Waltham, MA) overnight and 1:500 or 1:1,000 diluted horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody. The immunoreactive bands were visualized with Supersignal West Pico, Femto, or Atto plus chemiluminescent substrate solutions (Thermo Fisher Scientific, Waltham, MA). The images were acquired with Chemidoc Imager (Biorad, Hercules, CA). The membrane then was stripped and reblotted for β actin protein (loading control). Major immunoreactive bands of calnexin, EMC1, histone H2A, LAMP1, and β actin were analyzed by densitometry using Image J program. Arbitrary densitometric units for immunoreactive calnexin, EMC1, histone H2A, and LAMP1 were normalized with those of β actin. The densitometric ratios for respective proteins were compared among study groups. Fold changes in respective proteins were determined (normalized values of protein in E. coli-challenged and vehicle- or SPA4 peptide-treated cells/normalized value of protein in unchallenged and vehicle-treated cells).

Levels of TNF-α cytokine

TNF-α levels were measured in cell-free supernatants as previously described (22). Levels of secreted TNF-α were normalized with total cellular protein content.

Statistics

Statistical significance was analyzed using the t test or one-way ANOVA with post hoc Fisher least significant difference analysis (Prism software, La Zolla, CA). Statistical significance was defined as a p value of <0.05 or otherwise indicated.

RESULTS

The biochemical (oxidase negative, catalase positive) and growth characteristics of E. coli were maintained throughout the study. All batches of SPA4 peptide and FITC-SPA4 peptide were confirmed for their purity and composition by HPLC and mass spectrometry.

SPA4 peptide does not bind to E. coli and does not affect the bacterial growth in culture medium

Full-length native SP-A binds to a number of pulmonary pathogens, including Gram-negative bacteria, and directly kills the pathogens by increasing membrane permeability (23). Thus, we assessed whether SPA4 peptide derived from C-terminal region of human SP-A mimics direct anti-bacterial function of SP-A. Direct binding of the FITC-SPA4 peptide to live bacterial cells was assessed by flow cytometry and confocal microscopy. Polymyxin B binds strongly to LPS expressed in cell walls of Gram-negative bacteria. Incubation of bacterial cells with Oregon green 514-conjugated polymyxin B caused a significant shift in the fluorescence peak in flow cytometric histograms of bacteria indicating binding of polymyxin B (Fig. 1A). However, no shift was observed when FITC-SPA4 peptide was incubated with live bacterial cells. Bright green fluorescence of Oregon green 514-conjugated polymyxin B bound to bacteria was also visible in confocal images (Fig. 1B). No fluorescence was observed for bacteria incubated with FITC-SPA4 peptide.

Fig 1.

Fig 1

Open in a new tab

The SPA4 peptide does not bind or directly kill live E. coli. The log-phase bacteria were incubated with 10, 50, 75, and 100 µM FITC-SPA4 peptide, washed, and analyzed for fluorescence. Oregon green 514-conjugated polymyxin B (75 µM) served as control. The fluorescence of bacteria cultured in the presence of fluorochrome-conjugated SPA4 peptide and polymyxin B was analyzed by flow cytometry (A). Representative confocal images are shown in (B). In a separate set of bacterial-killing assays, E. coli 19138 was cultured in bacteriologic medium in the presence of 1, 10, and 100 µg/mL SPA4 peptide, and OD was measured at 600 nm for a period of 17 h. Treatment with ampicillin (3.5 µg/mL) served as control. The time-dependent changes in OD values (mean ± standard error of measurement or SEM) are shown in (C). Results were confirmed by colony counts (CFU/mL) obtained at 17 h. The colony counts are provided within the figure. Results are from one representative of two experiments performed in triplicate on separate occasions.

We also measured the effect of SPA4 peptide on the growth of E. coli 19138. Mid-log phase bacteria were cultured in the presence of SPA4 peptide or vehicle. Addition of SPA4 peptide did not affect bacterial growth as measured by OD600 or colony counts after 17 h of culture (4–6 × 108 CFU, Fig. 1C). These results are consistent with no binding of SPA4 peptide to P. aeruginosa and no effect on bacterial growth in culture medium (6).

SPA4 peptide enhances uptake of E. coli and suppresses the released amounts of TNF-α

It is well established that recognition, uptake, and innate immune responses against Gram-negative bacterial pathogens and ligands are predominantly orchestrated by TLR4. Thus, we studied whether TLR4-interacting SPA4 peptide would affect the phagocytic uptake of E. coli. We utilized flow cytometry to assess the change in percentages of cells with phagocytosed bacteria followed by visualization of cells by microscopy in different study groups. Our results demonstrate that therapeutically administered SPA4 peptide significantly increases the percentage of cells with phagocytosed GFP-expressing E. coli (Fig. 2). Furthermore, we observed that the bacterial uptake was increased in presence of a combination of SPA4 peptide and serum as compared to the cells treated with serum alone (p = 0.08, Fig. 2B). These results suggest pro-phagocytic activity of SPA4 peptide is not dependent on complement or other factors present in serum. Consistent to these findings, localization of pHrodo-conjugated E. coli inside the acidic phagolysosomes was also significantly increased in SPA4 peptide-treated cells (p < 0.0001, Fig. 3B and C). While tuftsin stimulates phagocytosis, cytochalasin D suppresses the bacterial uptake; both tuftsin and cytochalasin D were included as controls. Simultaneously, there was a significant reduction in secreted amounts of TNF-α in cell-free supernatants of E. coli-challenged and SPA4 peptide-treated cells (p < 0.05, Fig. 3D). The unstimulated JAWS II cells did not secrete detectable amounts of TNF-α.

Fig 2.

Fig 2

Open in a new tab

SPA4 peptide induces phagocytosis of live GFP-expressing E. coli 19138 (GFP-E. coli). The JAWS II cells were challenged with GFP-E. coli and treated with 75 µM SPA4 peptide and/or 1% autologous mouse serum. The cells were briefly washed and analyzed for GFP fluorescence by flow cytometry and confocal microscopy. The cells were gated for wide forward (FSC) and side scatter (SSC; shown in P region) and analyzed for GFP-associated fluorescence in FL1 channel. The unchallenged, untreated JAWS II cells (cells only control) gated in P1 region indicate no GFP fluorescence. The cells with phagocytosed GFP-E. coli were identified in P1 region among study groups. Representative flow cytometric charts are shown in (A). The cells with phagocytosed GFP-E. coli were noted as percent of cells in P1 region and compared with untreated cells incubated with GFP-E. coli. The bar chart shows data points and mean (± SEM) percent phagocytosis of GFP-E. coli as compared to basal phagocytosis from six experiments performed on separate occasions. (B, i). The mean fluorescent intensity of the cells among study groups is also shown (B, ii). The cells were then permeabilized, stained with nuclear dye (blue), and mounted with Vectashield for confocal microscopy. All the images were acquired with 63× objective under confocal microscope. Images taken at brightfield, and fluorescence channels were superimposed. Green fluorescence is of GFP-E. coli, and blue staining is of cell nuclei. Representative confocal microscopic images for each study group are shown in (C). The cells with phagocytosed GFP-E. coli were manually counted within nuclear plane. The bar chart shows data points and mean (± SEM) percent cells with phagocytosed GFP-E. coli in three to five different separate fields of confocal photomicrographs taken from two experiments (D, i). Enumerated bacteria per cell among study groups are shown in (D, ii). The p values are shown within the figures (t-test).

Fig 3.

Fig 3

Open in a new tab

SPA4 peptide treatment induces localization of pHrodo-conjugated E. coli K12 (pHrodo-E. coli) inside acidic phagolysosomes of dendritic cells but suppresses the TNF-α response. The time schedule of the experimental set up and respective assessments is shown in (A). The bar chart shows percent localization (data points and mean ± SEM) of pHrodo-E. coli in acidic phagolysosomes of dendritic cells after treatment with 75 µM SPA4 peptide. Tuftsin and cytochalasin D were included as positive and negative controls, respectively (B). Representative confocal microscopic images show localization of red fluorescent pHrodo-E. coli inside the acidic phagolysosomes of dendritic cells. The cell nucleus stained with Hoechst 33342 dye is shown in blue (C). Secreted levels of TNF-α cytokine were measured in cell-free supernatants of cells challenged with pHrodo-E. coli and treated with SPA4 peptide (D). Results are from five experiments performed on separate occasions. The p values are shown within each figure for statistical significance (t-test).

SPA4 peptide treatment augments the TLR4-dependent uptake of bacteria in the phagolysosomes and induces co-localization of TLR4 with bacteria

As expected, the phagocytic uptake of E. coli was increased in cells transfected with wild-type TLR4 (Fig. 4A); the SPA4 peptide treatment further augmented the TLR4-induced bacterial uptake. We observed about 30% increase in bacterial uptake in SPA4-treated cells overexpressing TLR4 compared to the cells transfected with vector plasmid DNA and treated with SPA4 peptide (p = 0.1, Fig. 4A). However, the bacterial uptake was not affected in SPA4 peptide-treated cells expressing dominant negative TLR4 (P→H mutation; Fig. 4B).

Fig 4.

Fig 4

Open in a new tab

SPA4 peptide augments the TLR4-dependent uptake and localization of pHrodo-conjugated E. coli K12 (pHrodo-E. coli) in phagolysosomes. The JAWS II dendritic cells were transfected with pDisplay vector plasmid DNA or with plasmid DNA encoding wild-type mouse TLR4 (A) to achieve overexpression of TLR4, challenged with bacteria, and treated with 75 µM SPA4 peptide. Similar experiments were performed with cells transfected with pDisplay vector plasmid DNA or with plasmid DNA encoding dominant negative mouse TLR4; dashed line represents basal phagocytosis in cells transfected with pDisplay vector plasmid DNA (B). Red fluorescence due to internalized bacteria was quantitated by fluorometry, and percent localization of bacteria was calculated relative to control; bars represent mean ± SEM of results from three separate experiments. Individual data points are shown within the figure. The p values are shown within each figure for statistical significance (t-test).

We also evaluated the intracellular and cell surface expression of TLR4 (green fluorescence) in cells challenged with pHrodo-E. coli and treated with SPA4 peptide or vehicle. No change was observed in overall expression of TLR4 in SPA4 peptide-treated cells in comparison with vehicle treatment at any time (Fig. 5B), except that the expression of TLR4 was slightly reduced and was visualized predominantly on the cell surface of dendritic cells at later time points after SPA4 peptide treatment (Fig. 5B and C).

Fig 5.

Fig 5

Open in a new tab

SPA4 peptide induces co-localization of TLR4 with pHrodo-conjugated E. coli K12 (pHrodo-E. coli) at early time point without affecting expression of TLR4 at any time (B, C). The bacterial challenge and treatment schedule are shown in (A). Representative images show the TLR4 expression and co-localization of TLR4 and pHrodo-E. coli in vehicle- and SPA4 peptide-treated cells at 2.5 and 4.5 h (C, i). The arrowhead (►) and asterisk (*) symbols depict the cell surface and intracellular expression of TLR4, respectively. Approximately 100 cells were analyzed in three to seven different fields of confocal micrographs from two to four experiments. The bar charts show percent cells expressing TLR4 (B) and co-localization of TLR4 with bacteria in each group (C, ii). The TLR4 staining in unchallenged, vehicle-treated cells is shown in (D). The p values are shown within the figure (t-test).

The co-localization (in yellow) of TLR4 (green fluorescence) and bacteria (red fluorescence) was noted in mid-nuclear plane in a blinded fashion. The intracellular co-localization of TLR4 with pHrodo-E. coli inside the phagolysosomes was enhanced after 1 h of treatment with SPA4 peptide (77%) in comparison to vehicle treatment (59%; p < 0.05, Fig. 5C). The co-localization of TLR4 with bacteria was observed in 80% of cells after 2 h of SPA4 peptide treatment and in 73% of cells at later time point (not statistically significant from those treated with vehicle, Fig. 5C). Co-localization on cell surface was always noted in less than 20% of cells treated with SPA4 peptide or vehicle.

Co-localization of ER proteins (calnexin and EMC1) with LAMP1 in SPA4 peptide-treated cells correspond with increased phagocytic uptake of E. coli

Consistently in different sets of experiments, we observed an increased bacterial uptake and localization of bacteria inside the acidic phagolysosomes in SPA4 peptide-treated cells (Fig. 3 and 5). Similar to calnexin, the EMC1 showed a punctate uniform staining throughout the cell cytoplasm. The SPA4 peptide treatment significantly increases the co-localization of calnexin protein with LAMP1 against challenge with heat-killed E. coli 19138 (66% versus 10% in vehicle-treated cells, p < 0.0001, Fig. 6B and F). Although co-localization of the EMC1 with LAMP1 was observed in fewer cells, it was significantly increased after SPA4 peptide treatment (41% versus 20% in vehicle-treated cells, p < 0.05, Fig. 6C and G).

Fig 6.

Fig 6

Open in a new tab

SPA4 peptide induces co-localization of ER-proteins: calnexin and EMC1 with LAMP1 (phagolysosome marker) against challenge with heat-killed E. coli 19138 at 2.5 h. The schedule of infectious challenge and treatment is shown in (A). Representative images show the SPA4 peptide-induced intracellular co-localization of calnexin and EMC1 with LAMP1 against challenge with heat-killed E. coli (B, C). Representative images of unchallenged cells, treated with vehicle or SPA4 peptide, and stained for calnexin or EMC1 and LAMP1 are also shown (D, E). Bar charts show the percent number of cells showing co-localization of calnexin and EMC1 with LAMP1 (F-I). Results are from four experimental wells for each group set up on separate occasions. The p values are shown within the figure (t-test).

Changes in expression of ER proteins (calnexin and EMC1), phagolysosome marker (LAMP1), and histone H2A protein

The immunoreactive protein bands of ~68, 70–75, and 90–100 kDa for calnexin, ~70 and 110 kDa for EMC1, ~120 kDa for LAMP1, and a single band between 15 and 20 kDa for histone H2A were identified in cell lysates after immunoblotting with antibodies specific to respective proteins. The expression of calnexin, EMC1, LAMP1, and histone H2A was studied at 1 h of SPA4 peptide treatment. No changes or only moderate changes were observed in the cellular expression of EMC1 and LAMP1 proteins, respectively (Fig. 7B, C, and D, ii and iii). The cellular expression of calnexin was slightly increased in SPA4 peptide-treated cells (20%, statistically not significant) compared to vehicle-treated cells against E. coli (Fig. 7B, C, and D, i) . The SPA4 peptide treatment significantly reduced the cellular expression of histone H2A proteins (~50%, densitometric ratio p = 0.0588, fold change p < 0.05, Fig. 7B, C, and D, iv) against E. coli.

Fig 7.

Fig 7

Open in a new tab

Assessment of expression of calnexin, EMC1, LAMP1, and histone H2A in cells challenged with heat-killed E. coli 19138 and treated with SPA4 peptide. The timeline of infectious challenge and SPA4 peptide treatment is shown in (A). The whole-cell lysate protein (2–5 µg) was separated and immunoblotted with antibodies specific to calnexin, EMC1, LAMP1, and histone H2A proteins. The membrane then was stripped and re-probed for β actin. Lanes contained whole-cell lysate protein from unchallenged, vehicle-treated control (1), heat-killed E. coli-challenged, vehicle-treated (2), and heat-killed E. coli-challenged, SPA4 peptide-treated (3) JAWS II dendritic cells. Representative immunoblots for each protein are shown within the figure (B, i–iv). The densitometric units for immunoreactive bands were normalized with those of β actin. The ratios of arbitrary densitometric units are shown as bar (Mean ± SEM) and are derived from three experiments performed on separate occasions (C, i–iv). The fold changes (calculated as normalized value for respective protein in experimental group per unchallenged, vehicle control group) are shown as bar (Mean ± SEM) in (D, i–iv). The p values are shown within each figure (one-way ANOVA).

SPA4 peptide treatment reduces secreted levels of TNF-α through its interaction with TLR4

The levels of TNF-α were significantly decreased in cell-free supernatants of SPA4 peptide-treated JAWS II cells (p < 0.05, Fig. 3) and in cells overexpressing TLR4 (p < 0.05, Fig. 8A). The TNF-α secretion was not affected in SPA4 peptide-treated cells expressing dominant negative TLR4 (Fig. 8B). Undetectable or only negligible amounts of TNF-α were detected in unchallenged cells transfected with vector plasmid DNA (3.8 pg/µg protein) or with plasmid DNA encoding dominant negative TLR4 (0 pg/µg protein) or wild-type TLR4 (4.2 pg/µg protein).

Fig 8.

Fig 8

Open in a new tab

SPA4 peptide suppresses TLR4-dependent secretion of TNF-α. The challenge with pHrodo-conjugated E. coli K12 (pHrodo-E. coli) and SPA4 peptide treatment of JAWS II dendritic cells transfected with pDisplay vector plasmid DNA or plasmid DNAs encoding wild-type (A) or dominant negative (B) mouse TLR4 were like those described in Fig. 4. TNF-α levels were measured in cell-free supernatants by ELISA and normalized with total cellular protein. TNF-α results presented here are from three to five experiments. The p values are shown within each figure (t-test).

DISCUSSION

The SPA4 peptide derived from the C-terminal region of human SP-A interacts with the TLR4 and exerts pro-phagocytic and anti-inflammatory activity against P. aeruginosa PAO1 (6). However, immunomodulatory activity of SPA4 peptide remained unknown against other Gram-negative bacteria. In this study, our results demonstrate pro-phagocytic and anti-inflammatory activity of SPA4 peptide against E. coli 19138 (a clinical isolate) and E. coli K12 through its interaction with TLR4 (Fig. 2 to 4). Furthermore, the overexpression of TLR4 in JAWS II cells augments the SPA4 peptide activity against E. coli (Fig. 4 and 8). The SPA4 peptide treatment did not affect the phagocytic or inflammatory response in JAWS II cells expressing dominant negative form of TLR4. An independent research group has previously reported that the phagocytic activity is significantly reduced in HEK293 cells expressing dominant negative TLR4 (24). Also, consistent with our previously published results (6), we observed a significant increase in fluorescence of phagocytosed pHrodo-conjugated E. coli in acidic phagolysosomes. An increased intracellular co-localization of TLR4 with E. coli substantiates the TLR4-modulatory activity of SPA4 peptide. As expected, the co-localization of TLR4 is specific against Gram-negative bacteria because TLR4 does not accumulate with phagocytosed Staphylococcus aureus (25). Our results also show pronounced co-localization of TLR4 with bacteria at early time point (Fig. 5). Dynamic synthesis and transfer process of TLR4 between cytoplasm and plasma membrane have been discussed in the literature (26), but not much is known about the fate of TLR4 associated with bacteria/ligand or phagolysosome. Along with other subcellular organelles, the ER could be involved at initial and late stages of bacterial phagocytosis, phagosome/phagolysosome formation, and TLR4 trafficking (10). We observed an increased expression and co-localization of calnexin with LAMP1 (Fig. 7) at an early time point suggesting the role of ER in bacterial uptake, lysis, and processing. An increased recruitment of calnexin with phagosomes has been shown after uptake of intracellular Legionella pneumophila (27) and Salmonella species (28). Our results suggest for the first time that the ER could be involved in pro-phagocytic activity of SPA4 peptide against Gram-negative bacteria.

Although the mechanism of ER membrane biogenesis, fusion with phagolysosome, and engagement of the ER with phagocytosed TLR4 and bacteria are not fully understood, the phosphoinsitol-3-kinase (PI3K) has been shown to regulate ER involvement in phagocytosis (12). The TLR4 is well recognized for its interaction with PI3K-AKT signaling (29). The ER proteins including EMC have been recognized as critical in the phagocytic process and membrane protein trafficking (12, 30). The EMC complex located in the ER is predicted to contribute to membrane insertase activity for protein insertion into the ER membrane. An increased co-localization of ER proteins (calnexin and EMC1) corroborates with recently published findings about the involvement of ER-calnexin in phagosome formation (12) and EMC1 in phagocytosis of Histoplasma capsulatum (14). The EMC1 is co-localized with phagolysosome marker LAMP1 in SPA4 peptide-treated cells, but the expression of EMC1 is not affected (Fig. 7). While dynamic relationship between expression and activity of EMC1 cannot be interpreted from the results presented here, it is likely that the EMC1 is involved in processing the phagocytosed bacteria and trafficking of membrane TLR4 due to its unique structure and enzymatic activity. These assumptions are based on published reports about emerging concept of the EMC serving as a central player in membrane protein biogenesis (31, 32). Detailed investigation is warranted in future to fully elucidate the mechanism of action of SPA4 peptide.

An overloading of proteins in the ER leads to ER stress and activation of inflammatory signaling (33). Our published work has established that the SPA4 peptide suppresses TLR4-MYD88-dependent NF-κB and AP-1, inflammatory cytokine and chemokine response, and inflammasome against LPS and Gram-negative bacterial stimuli (6, 7, 9, 21, 34, 35). The TLR4 stimulation of AP-1 and NF-κB transcription factors coincides with nucleosome (involving histones) and transcriptional activation of inflammatory response against infectious stimuli (15). Consistent with the previously published report that E. coli LPS-induced gene expression of histone cluster 2 and SPA4 peptide significantly suppresses the LPS-induced histone cluster 2 protein (7), we found that the expression of histone H2A protein was significantly reduced in SPA4 peptide-treated cells against E. coli (Fig. 7). Our results are consistent with previously published findings that the LPS induces the release of nuclear histone H2A into the cytoplasm (36). Suppression of histone H2A protein in SPA4 peptide-treated cells corroborates with the reduced TNF-α cytokine response (Fig. 3).

As reported for SP-A (37), the activity of SPA4 peptide may vary according to the bacterial characteristics. However, the pro-phagocytic and anti-inflammatory activity of SPA4 peptide remained consistent against P. aeruginosa PAO1 and E. coli K12 or 19138 strains, a comprehensive study is warranted to further investigate the immunomodulatory effects of the SPA4 peptide against multidrug-resistant strains of E. coli, P. aeruginosa, and other Gram-negative bacteria with different cell wall characteristics and LPS structures.

While antibiotic therapy is frequently successful in directly killing the pathogens with diverse characteristics and pathogenesis, clinical management becomes complicated in patients suffering from antibiotic-resistant Gram-negative bacterial infections. Synergistic activity of SP-A has been reported with polymyxin B (37). Thus, it will be of interest to investigate any synergistic activity of SPA4 peptide with colistin or polymyxin B used as last-choice treatments for antibiotic-resistant Gram-negative bacterial infections. It is also important to recognize that even though bacterial clearance is achieved, accompanying inflammation and end-organ damage can cause significant morbidity and mortality in many patients. Acute respiratory distress syndrome (ARDS) secondary to lung infection is one such example where lung injury and inflammation worsen the outcomes. While many pharmacotherapies have been tried, none has shown efficacy for patients with ARDS. The weakness of these therapies such as corticosteroids appears to be that while they inhibit inflammation, they also suppress antimicrobial host defense. Early trials of corticosteroids in ARDS increased the rate of infections. More recent trials using lower doses of corticosteroids demonstrated an improvement in clinical parameters but no effect on mortality (38–40). These outcomes may be associated with corticosteroid-mediated compromised immune cell functions, including phagocytosis and clearance of pathogens or ligands (41–43). An agent which promotes the phagocytic effect or pathogen clearance but simultaneously suppresses an inflammatory response may provide a better option for the treatment of ARDS. In this regard, an attractive target for immunomodulation is TLR4. Several TLR4-immunomodulators are currently being developed as a way mainly to control the inflammation during sepsis (44, 45). The SPA4 peptide interaction with TLR4 and its resultant dual pro-phagocytic and anti-inflammatory effects could provide an advantage over other TLR4-immunomodulators that are currently being developed or are under clinical trials (45). Anti-bacterial effects of these molecules are yet unknown. While the TLR4-interacting SPA4 peptide lacks the direct bacterial-killing function of native full-length SP-A (23), our results demonstrate the pro-phagocytic and anti-inflammatory activity of SPA4 peptide through its interaction with TLR4 reduce bacterial burden and inflammation, and alleviate the symptoms of sickness in a mouse model of bacterial lung infection (6). Detailed studies are being planned to investigate the synergistic activity in combination with current treatments and practices, mechanism of action, and translational potential of SPA4 peptide against Gram-negative bacterial infection and inflammation.

ACKNOWLEDGMENTS

Authors acknowledge technical help from Neha Chataut with microscopic analysis. Authors thank Dr. Nathan Shankar, Department of Pharmaceutical Sciences, College of Pharmacy, OUHSC, OK for providing the plasmid DNA encoding GFP. We also thank Dr. Mary Carter, Writing Center, OUHSC, OK, for providing editorial assistance.

Research reported here was supported by awards from an American Heart Association Grant-in-Aid (award number 11GRNT7220012) and National Heart, Lung, and Blood Institute of the National Institute of Health (award number R01 HL136325). The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Heart Association or the National Institute of Health.

B.S. performed phagocytosis assays and assessed direct binding of SPA4 peptide to bacteria. V.R. performed genetic transfection with TLR4 and experiments with pHrodo-conjugated E. coli, and measured TNF-α levels. N.M.G. performed immunocytochemistry, confocal microscopy, and immunoblotting. C.K. performed bacterial growth assays. S.A. designed and coordinated the study and compiled the manuscript.

Contributor Information

Shanjana Awasthi, Email: shanjana-awasthi@ouhsc.edu.

Kimberly A. Kline, Universite de Geneve, Geneva, Switzerland

REFERENCES

  • 1. Mancuso G, Midiri A, Gerace E, Biondo C. 2021. Bacterial antibiotic resistance: the most critical pathogens. Pathogens 10:1310. doi: 10.3390/pathogens10101310 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Almeida MC, da Costa PM, Sousa E, Resende DISP. 2023. Emerging target-directed approaches for the treatment and diagnosis of microbial infections. J Med Chem 66:32–70. doi: 10.1021/acs.jmedchem.2c01212 [DOI] [PubMed] [Google Scholar]
  • 3. Chiang MC, Chern E. 2022. More than antibiotics: latest therapeutics in the treatment and prevention of ocular surface infections. J Clin Med 11:4195. doi: 10.3390/jcm11144195 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Íñigo M, Del Pozo JL. 2022. Treatment of infections caused by carbapenemase-producing Enterobacterales. Rev Esp Quimioter 35 Suppl 3:46–50. doi: 10.37201/req/s03.11.2022 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Awasthi S, Madhusoodhanan R, Wolf R. 2011. Surfactant protein-A and toll-like receptor-4 modulate immune functions of preterm baboon lung dendritic cell precursor cells. Cell Immunol 268:87–96. doi: 10.1016/j.cellimm.2011.02.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Awasthi S, Singh B, Ramani V, Xie J, Kosanke S. 2019. TLR4-interacting SPA4 peptide improves host defense and alleviates tissue injury in a mouse model of Pseudomonas aeruginosa lung infection. PLoS One 14:e0210979. doi: 10.1371/journal.pone.0210979 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Awasthi S, Kumar G, Ramani V, Awasthi V, Rodgers KK, Xie J, Beierle J, Kyere-Davies G, Singh B, Rahman N, Chowdhury AA, Chataut N. 2021. Mechanism of anti-inflammatory activity of TLR4-interacting SPA4 peptide. Immunohorizons 5:659–674. doi: 10.4049/immunohorizons.2100067 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Chowdhury AA, Godbole NM, Chataut N, Kosanke S, Rodgers K, Awasthi S. 2022. Effects of SPA4 peptide on lipopolysaccharide-disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury. Physiol Rep 10:e15353. doi: 10.14814/phy2.15353 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Ramani V, Madhusoodhanan R, Kosanke S, Awasthi S. 2013. A TLR4-interacting SPA4 peptide inhibits LPS-induced lung inflammation. Innate Immun 19:596–610. doi: 10.1177/1753425912474851 [DOI] [PubMed] [Google Scholar]
  • 10. Ciesielska A, Matyjek M, Kwiatkowska K. 2021. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci 78:1233–1261. doi: 10.1007/s00018-020-03656-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Mantegazza AR, Magalhaes JG, Amigorena S, Marks MS. 2013. Presentation of phagocytosed antigens by MHC class I and II. Traffic 14:135–152. doi: 10.1111/tra.12026 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH, Steele-Mortimer O, Paiement J, Bergeron JJM, Desjardins M. 2002. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110:119–131. doi: 10.1016/s0092-8674(02)00797-3 [DOI] [PubMed] [Google Scholar]
  • 13. Volkmar N, Christianson JC. 2020. Squaring the EMC - how promoting membrane protein biogenesis impacts cellular functions and organismal homeostasis. J Cell Sci 133:jcs243519. doi: 10.1242/jcs.243519 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Cohen A, Jeng EE, Voorhies M, Symington J, Ali N, Rodriguez RA, Bassik MC, Sil A. 2022. Genome-scale CRISPR screening reveals that C3aR signaling is critical for rapid capture of fungi by macrophages. PLoS Pathog 18:e1010237. doi: 10.1371/journal.ppat.1010237 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Calao M, Burny A, Quivy V, Dekoninck A, Van Lint C. 2008. A pervasive role of histone acetyltransferases and deacetylases in an NF-kappaB-signaling code. Trends Biochem Sci 33:339–349. doi: 10.1016/j.tibs.2008.04.015 [DOI] [PubMed] [Google Scholar]
  • 16. Mizgerd JP, Lupa MM, Hjoberg J, Vallone JC, Warren HB, Butler JP, Silverman ES. 2004. Roles for early response cytokines during Escherichia coli pneumonia revealed by mice with combined deficiencies of all signaling receptors for TNF and IL-1. Am J Physiol Lung Cell Mol Physiol 286:L1302–L1310. doi: 10.1152/ajplung.00353.2003 [DOI] [PubMed] [Google Scholar]
  • 17. Crameri A, Whitehorn EA, Tate E, Stemmer WP. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14:315–319. doi: 10.1038/nbt0396-315 [DOI] [PubMed] [Google Scholar]
  • 18. Underhill DM, Goodridge HS. 2012. Information processing during phagocytosis. Nat Rev Immunol 12:492–502. doi: 10.1038/nri3244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Benincasa M, Pacor S, Gennaro R, Scocchi M. 2009. Rapid and reliable detection of antimicrobial peptide penetration into gram-negative bacteria based on fluorescence quenching. Antimicrob Agents Chemother 53:3501–3504. doi: 10.1128/AAC.01620-08 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Domingues MM, Inácio RG, Raimundo JM, Martins M, Castanho MARB, Santos NC. 2012. Biophysical characterization of polymyxin B interaction with LPS aggregates and membrane model systems. Biopolymers 98:338–344. doi: 10.1002/bip.22095 [DOI] [PubMed] [Google Scholar]
  • 21. Ramani V, Awasthi S. 2015. Toll-like receptor 4-interacting SPA4 peptide suppresses the NLRP3 Inflammasome in response to LPS and ATP stimuli. J Leukoc Biol 98:1037–1048. doi: 10.1189/jlb.3A1114-570R [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Vilekar P, Awasthi S, Natarajan A, Anant S, Awasthi V. 2012. EF24 suppresses maturation and inflammatory response in dendritic cells. Int Immunol 24:455–464. doi: 10.1093/intimm/dxr121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Wu H, Kuzmenko A, Wan S, Schaffer L, Weiss A, Fisher JH, Kim KS, McCormack FX. 2003. Surfactant proteins A and D inhibit the growth of gram-negative bacteria by increasing membrane permeability. J Clin Invest 111:1589–1602. doi: 10.1172/JCI16889 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Neal MD, Leaphart C, Levy R, Prince J, Billiar TR, Watkins S, Li J, Cetin S, Ford H, Schreiber A, Hackam DJ. 2006. Enterocyte TLR4 mediates phagocytosis and translocation of bacteria across the intestinal barrier. J Immunol 176:3070–3079. doi: 10.4049/jimmunol.176.5.3070 [DOI] [PubMed] [Google Scholar]
  • 25. Husebye H, Aune MH, Stenvik J, Samstad E, Skjeldal F, Halaas O, Nilsen NJ, Stenmark H, Latz E, Lien E, Mollnes TE, Bakke O, Espevik T. 2010. The Rab11A GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes. Immunity 33:583–596. doi: 10.1016/j.immuni.2010.09.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Klein DCG, Skjesol A, Kers-Rebel ED, Sherstova T, Sporsheim B, Egeberg KW, Stokke BT, Espevik T, Husebye H. 2015. CD14, TLR4 and TRAM show different trafficking dynamics during LPS stimulation. Traffic 16:677–690. doi: 10.1111/tra.12274 [DOI] [PubMed] [Google Scholar]
  • 27. Hubber A, Arasaki K, Nakatsu F, Hardiman C, Lambright D, De Camilli P, Nagai H, Roy CR. 2014. The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins. PLoS Pathog 10:e1004222. doi: 10.1371/journal.ppat.1004222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Santos JC, Duchateau M, Fredlund J, Weiner A, Mallet A, Schmitt C, Matondo M, Hourdel V, Chamot-Rooke J, Enninga J. 2015. The COPII complex and lysosomal VAMP7 determine intracellular Salmonella localization and growth. Cell Microbiol 17:1699–1720. doi: 10.1111/cmi.12475 [DOI] [PubMed] [Google Scholar]
  • 29. Laird MHW, Rhee SH, Perkins DJ, Medvedev AE, Piao W, Fenton MJ, Vogel SN. 2009. TLR4/MYD88/PI3K interactions regulate TLR4 signaling. J Leukoc Biol 85:966–977. doi: 10.1189/jlb.1208763 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Saitoh S-I. 2009. Chaperones and transport proteins regulate TLR4 trafficking and activation. Immunobiology 214:594–600. doi: 10.1016/j.imbio.2008.11.012 [DOI] [PubMed] [Google Scholar]
  • 31. Miller-Vedam LE, Bräuning B, Popova KD, Schirle Oakdale NT, Bonnar JL, Prabu JR, Boydston EA, Sevillano N, Shurtleff MJ, Stroud RM, Craik CS, Schulman BA, Frost A, Weissman JS. 2020. Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients. Elife 9:e62611. doi: 10.7554/eLife.62611 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Hegde RS. 2022. The function, structure, and origins of the ER membrane protein complex. Annu Rev Biochem 91:651–678. doi: 10.1146/annurev-biochem-032620-104553 [DOI] [PubMed] [Google Scholar]
  • 33. Pahl HL, Baeuerle PA. 1995. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factor NF-kappa B. EMBO J 14:2580–2588. doi: 10.1002/j.1460-2075.1995.tb07256.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. Madhusoodhanan R, Moriasi C, Ramani V, Anant S, Awasthi S. 2012. A TLR4-interacting peptide inhibits lipopolysaccharide-stimulated inflammatory responses, migration and invasion of colon cancer SW480 cells. Oncoimmunology 1:1495–1506. doi: 10.4161/onci.22089 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Awasthi S, Rahman N, Rui B, Kumar G, Awasthi V, Breshears M, Kosanke S. 2020. Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide. BMC Pulm Med 20:179. doi: 10.1186/s12890-020-01187-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Nair RR, Mazza D, Brambilla F, Gorzanelli A, Agresti A, Bianchi ME. 2018. LPS-challenged macrophages release microvesicles coated with histones. Front Immunol 9:1463. doi: 10.3389/fimmu.2018.01463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Coya JM, Fraile-Ágreda V, de Tapia L, García-Fojeda B, Sáenz A, Bengoechea JA, Kronqvist N, Johansson J, Casals C. 2022. Cooperative action of SP-A and its trimeric recombinant fragment with polymyxins against gram-negative respiratory bacteria. Front Immunol 13:927017. doi: 10.3389/fimmu.2022.927017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Meduri GU, Marik PE, Chrousos GP, Pastores SM, Arlt W, Beishuizen A, Bokhari F, Zaloga G, Annane D. 2008. Steroid treatment in ARDS: a critical appraisal of the ARDS network trial and the recent literature. Intensive Care Med 34:61–69. doi: 10.1007/s00134-007-0933-3 [DOI] [PubMed] [Google Scholar]
  • 39. Agarwal R, Nath A, Aggarwal AN, Gupta D. 2007. Do glucocorticoids decrease mortality in acute respiratory distress syndrome? A meta-analysis. Respirology 12:585–590. doi: 10.1111/j.1440-1843.2007.01060.x [DOI] [PubMed] [Google Scholar]
  • 40. Peter JV, John P, Graham PL, Moran JL, George IA, Bersten A. 2008. Corticosteroids in the prevention and treatment of acute respiratory distress syndrome (ARDS) in adults: meta-analysis. BMJ 336:1006–1009. doi: 10.1136/bmj.39537.939039.BE [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Mukherjee N, Mukherjee S, Saini P, Roy P, Sinha Babu SP. 2014. Antifilarial effects of polyphenol rich ethanolic extract from the leaves of Azadirachta indica through molecular and biochemical approaches describing reactive oxygen species (ROS) mediated apoptosis of Setaria cervi. Exp Parasitol 136:41–58. doi: 10.1016/j.exppara.2013.11.006 [DOI] [PubMed] [Google Scholar]
  • 42. Kemper MF, Stirone C, Krause DN, Duckles SP, Procaccio V. 2014. Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection. Eur J Pharmacol 723:322–329. doi: 10.1016/j.ejphar.2013.11.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Rosenberg JC, Lysz K. 1980. Suppression of the immune response by steroids. comparative potency of hydrocortisone, methylprednisolone, and dexamethasone. Transplantation 29:425–428. doi: 10.1097/00007890-198005000-00019 [DOI] [PubMed] [Google Scholar]
  • 44. Peri F, Piazza M. 2012. Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists. Biotechnol Adv 30:251–260. doi: 10.1016/j.biotechadv.2011.05.014 [DOI] [PubMed] [Google Scholar]
  • 45. Connolly DJ, O’Neill LAJ. 2012. New developments in Toll-like receptor targeted therapeutics. Curr Opin Pharmacol 12:510–518. doi: 10.1016/j.coph.2012.06.002 [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES