Shock/sepsis-induced acute lung injury (indirect-acute lung injury [iALI]) and its more severe form, acute respiratory distress syndrome, have high morbidity and mortality (1). Current interventions are limited to supportive therapy, including low tidal volume ventilation, as no real molecularly based therapeutic approach has yet become available (2). The pathophysiologic process of iALI includes immune cell accumulation, cytokine release, epithelial/endothelial cell injury, and inhibited neutrophil apoptosis (2). However, the detailed pathogenic mechanism is not yet clear.
Herpes virus entry mediator (HVEM), also known as tumor necrosis factor receptor superfamily-14 (TNFRSF14), is a transmembrane molecule expressed in nearly all internal organs. The highest expression of HVEM is in the lung, kidney, and liver. The cellular distribution of HVEM is relatively wide; it is found on T cells, B cells, dendritic cells, myeloid cells, epithelial cells, and endothelial cells (3). HVEM can engage up to five different ligands (CD160, B and T lymphocyte attenuator [BTLA], LIGHT [tumor necrosis factor superfamily member 14, TNFSF14], lymphotoxin α, and herpes simplex virus glycoprotein D) to deliver inhibitory or stimulatory signals (4). Studies have found that HVEM signaling activates intestinal epithelial cells to produce inflammatory cytokines that assist to provide host defense against pathogenic enteric bacteria (5, 6). However, the role of HVEM in the development of shock/sepsis-induced ALI in mice is not well studied. We hypothesize that HVEM signaling may activate pulmonary cells to promote inflammation in iALI. To test this hypothesis, we utilized a mouse model of combined shock (hemorrhage [HEM]) and sepsis (cecal ligation and puncture [CLP]) induced iALI, to investigate the relationship of pulmonary HVEM expression with local inflammation and lung injury.
MATERIALS AND METHODS
C57BL/6 male mice, 20 g to 25 g body weight, ages 8 to 12 weeks, were obtained from the Jackson Laboratory (Bar Harbor, Maine) and were housed in RI Hospital's Central Research Facilities for 7 to 10 days before experiments commenced. All protocols were carried out in the morning (8–11 AM) according to the National Institutes of Health guide for animal use and care, and were approved by the Lifespan-Rhode Island Hospital Institutional animal care and use committee (approval number: 0040-16). Animals were housed in ventilated racks, with water and fed standard mouse chow ad libitum under standard environmental conditions (12-h: 12-h light/dark cycle, 68–72°F, 30%–70% humidity).
A mouse model of iALI was induced by HEM shock followed 24 h later by CLP septic challenge, as previously described by our laboratory (7, 8). After being anesthetized with isoflurane, mice were bled from one femoral artery to a mean blood pressure of 30 mm Hg to 40 mm Hg, kept stable for 90 min, and resuscitated with Ringers lactate (four times the shed volume). Sham mice incurred bilateral femoral artery ligated, but no blood was drawn. 24 h post-HEM or post-sham HEM, mice were subjected to a septic insult by CLP. The cecum was isolated and ligated at a point approximately 1 cm from the cecal tip, punctured twice with a 22-gauge needle, then gently squeezed to extrude a small amount of fecal material from the perforation sites. The midline wound was closed in two layers and mice were resuscitated with 1 mL of Ringers lactate subcutaneously. In the sham CLP group mice, the cecum was exposed but neither ligated nor punctured.
HVEM siRNA treatment
To confirm the efficacy of anti-HVEM silencing ribonucleic acid (siRNA) (SMART pool siGENOME Mouse Tnfrsf14 siRNA [M-058214-00-0050]) (GE Dharmacon, Lafayette, Colo), in vitro cell culture of mouse lung epithelial cell line-LA4, obtained from the American Type Culture Collection (Manassas, Va), were cultured with Dulbecco's Modified Eagle's Medium/F-12 media containing 10% fetal bovine serum according to manufacturer's directions. Cells were seeded in tissue culture plates until they reached 60% to 80% confluent. The culture media were replaced by the Opti-MEM media with Lipofestamine containing anti-HVEM siRNA (100 nM) or phosphate buffered saline (PBS) and cells were cultured for another 24 to 48 h. Cells were collected to determine HVEM expression by flow cytometry.
For in vivo mouse studies, animals were randomized to receive either anti-HVEM siRNA, at a dose of 50 μg in 100 μL PBS or 100 μL PBS per mouse, by intra-tracheal (I.T.) instillation 2 h after HEM (9). Of note, we have shown in prior studies that nonsense control siRNA, at the concentration used here, has little effect on indices of experimental iALI as produced by this model when compared with the PBS vehicle treatment (10–12). Therefore, we opted to compare these animals to a PBS alone control for these studies.
At 24 h post-CLP or sham CLP (48 h after Hem+CLP), mice were euthanized with an isoflurane overdose. Broncho-alveolar lavage fluid (BAL) was collected. Briefly, the trachea was exposed via a midline incision and cannulated with a sterile polypropylene 21-gauge catheter. The lungs were lavaged twice with 0.6 mL of normal saline. The lavage fluid was collected for cell count and protein concentration measurement. The whole lung tissue was harvested and homogenized for Western blot, cytokine, and myeloperoxidase (MPO) analyses as previously outlined (13) or in some cases the lungs were perfused with PBS, then enzymatically dissociated to produce a single-cell suspension for flow cytometric staining/analysis as described in detail by our laboratory before (14, 15).
Investigated proteins include HVEM (D-5) (Santa Cruz Biotechnology, Inc, Dallas, Tex), Phospho-signal transducer and activator of transcription (STAT)-3 (p-STAT-3) (Tyr705), STAT-3 (124H6), and Phospho-p65 (Ser536) antibodies (Cell Signaling Technology, Danvers, Mass). Corresponding secondary antibodies were used according to the manufacturer's protocol. Image densitometry was collected and analyzed with ImageJ software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or STAT-3 were used as a loading controls (Life Technologies) (16).
HVEM expression in the dissociated lung tissue cell populations or the LA-4 cell line was determined with PE-labeled anti-HVEM (clone HMHV-1B18) (Biolegend, San Diego, Calif), combined with antibodies to denote cell type: APC-labeled anti-Epcam (clone G8.8), anti-CD11c (clone N418), anti-B220 (clone RA3–6B2), anti-CD31 (clone 390), anti-Gr1 (clone 1A8-Ly6 g), anti-F4/80 (clone BM8), and anti-CD3 (clone SK7) antibodies (eBioscience, San Diego, Calif).
Measurement of cytokines
Concentrations of keratinocyte chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 from lung lysates were assessed via an enzyme-linked immunosorbent assay according to manufacturer's directions (BD Bioscience, San Jose, Calif).
Lung myeloperoxidase (MPO) activity
Lung tissue MPO activity was measured using an assay kit (ab105136) according to manufacturer's directions (Abcam, Cambridge, Mass).
Assessment of lung morphology
The left lung was inflated at 15 mm Hg and fixed with 10% formalin. The specimens were processed, stained with hematoxylin and eosin, and examined by light microscopy. The images were collected from the microscope (Nikon Eclipse 80i) using a ×20 objective and a Spot RT3 camera (13).
Both HVEM siRNA and PBS treated mice (n=15/group) were subjected to the combined insults of Hem/CLP, and were observed for survival over a 10-day period.
Results for all studies other than survival are expressed as mean ± standard error of the mean (SEM); n = 4–6 mice per group. Statistical significance of the results presented were determined by one-way analysis of variance (ANOVA) with Bonferroni post-hoc multiple comparisons test. Log-rank test was used for the survival study. Statistical software used was Graph Pad Prism 5 (La Jolla, Calif). P < 0.05 was used as a cutoff for establishing evidence of a significant difference between a given group.
HVEM expression is elevated in response to iALI
Following the establishment of our anti-HVEM siRNA SMART constructs capacity to selectively inhibit the expression of HVEM protein by the cultured mouse lung epithelial cell line-LA4 (Fig. 1A), we first sought to determine whether HVEM was localized in pulmonary cells during ALI. As shown in Figure 1B and C, HVEM is detected in lung tissue, and the expression is significantly elevated in iALI mice compared with sham mice. This suggests the possibility that HVEM expression may play a role in the development of iALI. By flow cytometry, we subsequently found that HVEM expression was upregulated significantly on Epcam+ (epithelial cell, Fig. 2A), CD11c+ (dendritic cell, Fig. 2B) and B220+ (B cell, Fig. 2C) cell populations. There was no statistically significant change of HVEM expression on CD31+ (endothelial cell, Fig. 2D), Gr1+ (neutrophil, Fig. 2E), F4/80+ (macrophage, Fig. 2F), and CD3+ (T cell, Fig. 2G) cells during iALI.
In previous studies (12, 17), we have shown that I.T. delivery of naked siRNA reduced target molecule expression primarily in pulmonary epithelial cells, but did not appear to markedly affect other cells. Inasmuch we initially chose to examine the impact the I.T. administration of naked anti-HVEM siRNA on the iALI-induced elevation of HVEM protein expression in the lung, as assessed by Western immunoblot analysis. We found that the I.T. administration of anti-HVEM siRNA markedly attenuated the rise in HVEM expression seen in the iALI (PBS) group (Fig. 1, A and B). With respect to the impact of I.T. anti-HVEM siRNA delivery on lung cell sub-population level, we further observed that HVEM was significantly decreased, specifically on Epcam+ cells (Fig. 2A). This effect was not found among other cell sub-populations, including CD11c+, B220+, CD31+, Gr1+, F4/80+, and CD3+ cells (Fig. 2, B–G).
To investigate which signaling pathways may be involved in HVEM expression, we measured phosphorylated STAT-3 in lung tissue lysates by Western blot. Phosphorylated STAT-3 was increased in iALI mice compared with sham mice, and decreased in HVEM-siRNA treated mice compared with the PBS treated iALI group (Fig. 3, A and B). There was no relationship between HVEM expression and p65 phosphorylation of the NFκB cascade (data not shown).
Knocking down HVEM in pulmonary epithelial cells with HVEM-specific siRNA attenuates lung inflammation and reduces early mortality after iALI.
Increased levels in inflammatory cytokines are thought to be indices of local pulmonary inflammation and injury (18). In the assessment of whether local pulmonary inflammation was affected by I.T. instillation of HVEM-siRNA into the lungs, especially relative to epithelial cells, we found that chemokines MCP-1 (Fig. 4A), MIP-2 (Fig. 4B), and KC (Fig. 4C) in lung lysates were increased significantly in mice subjected to iALI, and decreased in HVEM-siRNA treated iALI mice. This indicates that HVEM expression may affect immune cell recruitment to the lungs. In addition, IL-6 (Fig. 4D) levels were increased significantly in PBS treated iALI mice when compared with sham mice, and were decreased to sham levels with HVEM-siRNA treatment in iALI mice. However, there were no changes in IL-10 or TNF-α levels in lung tissue lysates between groups (Fig. 4, E and F).
MPO activity, which is used as a measure of neutrophil presence in lung tissue, was elevated in iALI mice compared with sham controls, and was decreased significantly with local silencing of HVEM (Fig. 5A).
Increased protein concentration in BAL is commonly used as a marker of vascular pulmonary permeability/lung injury (19). In this study, BAL protein concentration was significantly elevated in iALI mice when compared with sham controls, while it was decreased significantly in iALI mice treated with HVEM-siRNA compared with PBS treatment (Fig. 5B). Moreover, the increased total cell count of BAL seen in iALI mice declined in the HVEM-siRNA treated group (Fig. 5C).
Morphologically, paraffin-embedded sections of the lung stained with hematoxylin and eosin showed evidence of increased congestion and neutrophil influx in iALI mice when compared with sham controls. However, the HVEM-siRNA treated animals subjected to iALI appeared to have reduced lung inflammatory infiltrates and improved alveolar architecture, which was more similar to sham controls (Fig. 6A). Finally, the survival rate was elevated significantly in HVEM-siRNA treated group compared with PBS treated iALI mice during the first 60 h after CLP; however, this survival advantage was lost by 10 days (Fig. 6B).
The pathogenic mechanism of iALI is not yet clear (2). The function of HVEM in this disease state is to promote inflammation and to stimulate the clearance of bacteria as previously described (5, 20). Our study reveals that HVEM may play an important role in the development of iALI.
We observed that HVEM expression was elevated on multiple cell types in the lungs during iALI, including epithelial cells, dendritic cells, and B cells. Beyond the antigen-presenting cells, we also found an increased trend of HVEM expression on endothelial cells and decreased HVEM expression on neutrophils and T cells, a finding consistent with the presence of severe inflammation coexisting with dampened bacterial clearance in iALI. The treatment with naked HVEM-specific siRNA delivered by I.T. instillation has previously been documented to primarily affect epithelial cells (10, 12, 17, 21); thus, the following results could be partially taken as identifying a pathological role of HVEM signaling in pulmonary epithelial cells.
In the past, the quantity of neutrophils and macrophages in the lungs has been correlated with an ALI prognosis (22, 23). Here, we found that HVEM knock-down reduced pulmonary inflammation in our experimental model of iALI. As epithelial cells are reported to be one of the main sources of chemokines (24) and as I.T. instillation favors lung epithelial cells as a target (10, 12, 17), we chose to look at changes in lung chemokine levels. Downregulation of epithelial HVEM expression brought about a reduction of chemokine (MCP-1, KC, and MIP-2) levels. This in turn was associated with local decline in lung neutrophil numbers as well as BAL inflammatory cell numbers. This indicates that HVEM signaling through pulmonary epithelial cells, potentially via augmented chemokine release, etc., influences immune cell recruitment—one of the central links in iALI development (2, 21, 25).
Relative to pulmonary inflammation, levels of the pro-inflammatory cytokine IL-6 were reduced after HVEM gene knockdown. Consistent with the pro-inflammatory profile of the TNFR superfamily, HVEM has previously been shown to induce IL-6 secretion (5, 26). Additionally, the decreased local inflammatory cell influx (lower BAL cell count) into the lungs likely contributed to the suppressed levels of IL-6 in the anti-HVEM siRNA treated iALI mouse group. Another facet of this diminished IL-6 may have been the muted inflammatory response leading to a milder epithelial injury, which in turn incited a less vehement IL-6 production. This is not the case for TNF-α and IL-10, however, as these cytokines did not correlate with HVEM expression, implying that their regulation is independent of HVEM expression. It may also be that as delivery of naked siRNA construct I.T. appears to be taken up largely by pulmonary epithelial cells and not macrophage or endothelial cells (which are reported to be significant sources of TNF-α and/or IL-10 in this model) (12, 17). Therefore, the production of these cytokines may not have been affected much here. Increased BAL protein concentration is indicative of pulmonary vascular permeability/lung injury (27). We further observed that with anti-HVEM siRNA intervention, BAL protein levels were markedly reduced, suggesting the alleviation of pulmonary epithelial/endothelial barrier injury, consistent with our findings on histologic analysis.
The marked, although transient, improvement of survival rate seen here is likely the combined consequence of the above factors. We think that the nature of the transient survival benefit may be related to the inability of the single time-point administration of siRNA's capacity to sustain suppression of HVEM gene expression in high turnover cells like pulmonary epithelial cells, which we have shown to be a target of I.T. siRNA delivery (17). It is possible that if the delivery frequency were increased, the duration of the survival benefit might be improved further. With this stated, it must be appreciated that semiselective epithelial targeting of I.T. delivered siRNA may preclude targeting of other more distal cell/tissues/organs that are known to be affected by shock/sepsis (e.g., liver, kidney, intestine). The modulation of these organ systems following shock/sepsis contributes to overall mortality and warrants further exploration. We aim to examine this in future studies by assessing the impact of not only alternative routes of delivery, but also alternative modes of siRNA encapsulation.
HVEM has been reported to activate the NF-κβ and STAT-3 pathways (5, 28–30). In our study, the increase in iALI-induced HVEM expression correlated with STAT-3 phosphorylation, but not NFκB p65. However, as iALI-induced STAT-3 activation/phosphorylation was suppressed by I.T. delivery of anti-HVEM siRNA, this would support the epithelial cell-HVEM-STAT-3 mediated regulation of mucosal cell inflammation in this model, in line with what has been reported by others (5, 31). Yet, which specific pathway(s) HVEM utilizes to recruit/activate STAT-3 within the lung epithelial cells remains to be established. Beyond this, HVEM has the ability to promote cell-survival (32). The decreased neutrophil apoptosis, together with the increased epithelial/endothelial cells apoptosis, are thought to be important pathologic events in iALI (33, 34), and HVEM is expressed on all of these cell types. The type of influence that HVEM exerts on cellular apoptosis in this iALI model remains to be elucidated.
Finally, a number of questions remain unresolved that were outside the scope of our initial study design. What are the HVEM ligands that are expressed in response to insults associated with experimental iALI? Since HVEM has been reported to either enhance or prevent inflammation depending on its interaction with the potential ligands or receptors in different configurations (35), which ones might be pathologically dominant in the iALI lung? Do these ligands/receptors provide stimulatory or inhibitory signals, or both? Alternatively, as many of these HVEM ligands and receptors (e.g., LIGHT and BTLA) also transduce intracellular signals themselves when engaged by HVEM, what might this mean when translated into the murine Hem/CLP model? The initial findings here provide foundation for further investigation.
In conclusion, our study shows, for the first time, that HVEM plays an important role in experimental iALI. Further, anti-HVEM siRNA, when delivered intratracheally, can reduce inflammatory cell recruitment, mitigate local pulmonary inflammatory response, alleviate lung injury, and transiently reduce mortality induced by experimental iALI. The fact that HVEM is poised at the interconnection of several signaling pathways, many of which act bidirectionally, renders this molecule an interesting and potentially unique therapeutic target in iALI.
The authors thank Mr Paul Monfils and Ms Virginia Hovanesian, Core Research Laboratories, Rhode Island Hospital, for assistance with histology and microscopy/imagine analysis of these specimens.
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Keywords:© 2019 by the Shock Society
Co-inhibitory receptor/ligand; indirect lung injury; mouse; siRNA