s2fieldsofstudy: Biology Medicine

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Identification of a Subset of Microsatellite-Stable Endometrial Carcinoma with High PD-L1 and CD8+ Lymphocytes

Immune checkpoint blockade has emerged as an effective therapeutic strategy for patients with advanced cancer. Identification of biomarkers associated with treatment efficacy will help to select patients more likely to respond to this approach. High levels of microsatellite instability, tumor expression of PD-L1, high tumor mutation burden, and increased tumor infiltrating lymphocytes have all been associated with response to checkpoint inhibitor blockade. The purpose of this study was to determine if a subset of microsatellite stable endometrioid endometrial carcinomas have higher immune cell infiltrates and/or expression of PD-L1. PD-L1 expression and characterization of immune cell infiltrates were analyzed in 132 microsatellite stable, FIGO grade 2 endometrioid carcinomas. PD-L1 was positive in 48% (63/132) of the tumors. Tumor cell expression of PD-L1 was significantly associated with lymphatic/vascular invasion and deep myometrial invasion. PD-L1 expression was especially prominent at the invasive front and in foci of tumor-associated squamous metaplasia. Twenty-one cases (16% of the total) with more diffuse and/or especially strong PD-L1 expression were identified. This PD-L1 high subset was associated with significantly higher numbers of tumor-associated CD3+ and CD8+ lymphocytes. Only one tumor in the PD-L1 high subset harbored a POLE mutation. PTEN immunohistochemical loss, a common event in endometrioid-type endometrial carcinoma and associated with local immune suppression in melanoma, was not associated with PD-L1 expression or lymphocyte/macrophage infiltration of the tumor. These results suggest that a subset of microsatellite-stable endometrial cancers has higher expression of PD-L1 and increased tumor-associated CD3+ and CD8+ lymphocytes, characteristics more commonly associated with endometrial cancers with high levels of microsatellite instability. These results suggest that screening strategies to select only microsatellite instability-high advanced endometrial cancers for checkpoint inhibitor therapy might exclude patients who could potentially benefit from this therapeutic approach.

Introduction
Endometrial cancer can be classified into four molecular clusters, including the ultramutated (characterized by mutation in the gene polymerase epsilon, POLE), hypermutated (characterized by high levels of microsatellite instability), copy number low (characterized by absence of POLE and TP53 mutations and absence of microsatellite instability-high), and copy number high (characterized by presence of TP53 gene mutations) clusters (1). Two of these groups (POLE and microsatellite instability-high) are associated with a high predicted mutational load. The neoantigens generated by the high mutational load in these tumors have been shown to elicit a more robust host immune response (2,3). Both of these groups are associated with higher tumor-associated immune cells than the tumors in the microsatellitestable group (2)(3)(4)(5). Similarly, PD-L1 expression has been demonstrated to be higher in mismatch repair-deficient endometrial carcinomas compared to mismatch repair intact tumors (4)(5)(6). Therefore, much of the attention of immunotherapy research and clinical trials has focused on the microsatellite instability-high and POLE-mutated tumors (4)(5)(6)(7).
While microsatellite instability is routinely measured in many clinical pathology laboratories, POLE is not commonly interrogated by most clinically available nextgeneration sequencing hotspot panels. Quantifying tumor mutation burden typically requires whole exome sequencing or, at the very least, a sequencing panel of several hundred genes. Thus, most clinical labs lack effective means to assess tumor mutation burden. A complicating issue is that not all tumors with high mutation burdens have POLE mutation or high levels of microsatellite instability (8). For endometrial cancer, it was recently shown that elevated predicted neoantigen load was associated with better prognosis in the TCGA copy number low endometrioid group and in the copy number high serous-like group; neither of these groups is associated with POLE mutation or microsatellite instability-high (9). These results suggest that in addition to the POLE mutated and microsatellite instabilityhigh endometrial cancers, there are specific subsets of hypomutated endometrial cancer that may be more immunogenic and have improved outcomes and potentially better response to immunotherapy. Therefore, the purpose of the current study was to determine if a subset of microsatellite-stable/mismatch repair intact endometrial cancers exhibit increased tumor cell expression of PD-L1 expression and an immune microenvironment with increased numbers of tumor associated immune cells.

Patient Selection
The study was approved by the University of Texas MD Anderson Cancer Center's institutional review board (Protocol LAB01-718). Retrospective review from January 1, 2013 -May 5 th , 2016 revealed 382 consecutive cases of endometrial carcinoma with available tissue that had previously been tested for DNA mismatch repair protein immunohistochemistry and/or microsatellite instability. It has been shown that endometrial tumor histology can influence PD-L1 expression, with higher expression observed in grade 3 endometrioid and non-endometrioid histologies (10). To minimize any possible variation in immune cell infiltrate that could be attributed to the histotype or grade of tumor, we selected for further examination FIGO grade 2 endometrioid carcinomas, as this was the most common histotype. A total of 132 microsatellite-stable, FIGO grade 2 endometrial endometrioid cancers with intact immunohistochemical expression of MLH1, MSH2, MSH6, and PMS2 were identified for further study (Figure 1). Tumor histotype was verified by light microscopic examination of H&E stained slides. Clinical and pathological variables, including, patient age, tumor size, myometrial invasion (less than or greater than 50%), lymphovascular space invasion, and tumor stage, were derived from the electronic medical record and pathology reports.

Assessment of DNA mismatch repair
Mismatch repair protein and microsatellite instability testing were performed according to methodology previously described (11). Immunohistochemistry of mismatch repair proteins was performed using standard techniques for MLH1 (G168-15 1:25; BD Biosciences Pharmingen), MSH2 (FE11, 1:100; Calbiochem), MSH6 (44, 1:300; BD Biosciences Pharmingen), and PMS2 (Alb-4, 1:125; BD Biosciences Pharmingen). Immunohistochemistry was scored as mismatch repair protein intact or deficient using light microscopic examination. Complete absence of mismatch repair protein expression was required in order for a case to be designated as mismatch repair deficient. Stromal cells served as an internal positive control. PCR-based microsatellite instability analysis was performed using 7 microsatellites (TGFBR2 and the 6 National Cancer Institute recommended microsatellites, BAT25, BAT26, BAT40, D2S123, D5S346, and D17S250). A tumor was considered microsatellite instability-high if three or more of the seven markers demonstrated allelic shift. Tumors without allelic shift were designated as microsatellite stable.

Tumor-associated immune cell infiltrate analysis
Immunohistochemistry with antibodies for CD3 (lymphocytes), CD8 (lymphocytes), and CD68 (macrophages) was used to determine the composition, density, and distribution of the tumor-associated immune cell infiltrates. Immunohistochemical stained slides were analyzed using image analysis software (Aperio ImageScope) in order to quantify the number of positive cells within designated areas. Slides were scanned at 20X (Aperio Scanscope AT Turbo; Leica Biosystems) utilizing methods previously described (17). The areas of highest positive cell density for each immune cell marker were identified by placement of five 1 mm 2 boxes in the center of the tumor and along the periphery/leading edge of the tumor ( Figure 2). The periphery/leading edge of the tumor was defined as +/− 1 mm from the invasive tumor front (if present) or +/− 1 mm from the endometrial-myometrial interface. The center of the tumor was defined as the area greater than 1 mm towards the luminal aspect from the endometrial-myometrial interface or invasive tumor front ( Figure 2). Therefore, for both the tumor periphery and center, a total of 5 mm 2 areas containing the highest density of CD3-, CD8-, or CD68-positive cells were identified. The single highest density box among these was designated as the hotspot for each immune cell marker. "Overall" was defined as the combined sum of the positive cells tabulated along the tumor periphery and within the tumor center. For each region (periphery, center, hotspot, and overall), the number of total positive cells was added and divided by the total area (mm 2 ) in which the cells were counted. The results for CD3, CD8, and CD68 were reported as number of positive cells/mm 2 .

POLE Mutational Analysis
The cases designated as tumor PD-L1 high (n=21) were tested by Sanger sequencing for mutation in the exonuclease domain of DNA polymerase ε (POLE; exons 9-14) (18). Microdissected sections from formalin-fixed, paraffin-embedded tumor were used for sequencing. DNA was extracted using the Pico Pure DNA extraction kit (Applied Biosystems).

Statistical Analysis
Demographic and clinical characteristics for all patients were summarized with descriptive statistics. Characteristics were compared by tumor PD-L1 status (negative vs. positive; negative vs high positive). Wilcoxon rank-sum test was used to compare continuous variables, and chi -squared or Fisher's exact test was used to compare categorical variables. A Bonferroni correction was used to control the false discovery rate within each of the main comparisons. Statistical significance threshold was defined at 0.0025 (0.05/20). All statistical analyses were conducted using Stata v14.1 (College Station, TX).

Results
The clinical and pathological characteristics for the 132 cases of mismatch repair intact endometrial endometrioid adenocarcinoma, FIGO Grade 2, are summarized in Table 1. The median patient age was 60 years. The majority of patients presented with early stage disease (stage I or II, 89% of patients).
PD-L1 was positive in 48% (63/132) of the tumors. The majority of cases had weaker 1-2+ PD-L1 staining (55/63, 87%) and/or PD-L1 staining was very focal, staining 1% of the tumor (43/63, 68%). Tumor cell expression of PD-L1 was associated with deeper myometrial invasion and the presence of lymphatic/vascular space invasion in the hysterectomy ( Table 2). Both advanced tumor stage (p=0.012) and PTEN loss (p=0.012) showed trends towards being associated with PD-L1 expression, but these differences were not statistically significant given the threshold of 0.0025 to control for false discovery with multiple comparisons (Table 2).
Despite controlling for endometrial cancer histotype (endometrioid), grade (FIGO 2), and having comparable mean patient ages for PD-L1 negative and PD-L1 positive groups, a broad spectrum of CD3+, CD8+, and CD68+ expression within the tumor was encountered. In general, CD3+ or CD8+ lymphocytes comprised most of the tumor-associated immune cell infiltrate, with smaller numbers of CD68+ macrophages ( Table 2). The numbers of lymphocytes and macrophages did not differ significantly when comparing tumor center to tumor periphery (Table 2). Tumor cell expression of PD-L1 was not associated with significant differences in CD3+ or CD8+ tumor-associated lymphocytes, although the PD-L1 positive group showed trends towards higher numbers of these lymphocytes ( Table 2). The number of CD68+ tumor-associated macrophages did not differ significantly according to PD-L1 expression ( Table 2). Independent of PD-L1 expression, the presence of myometrial invasion, myometrial invasion ≥50% myometrial thickness, and lymphatic/ vascular space invasion were not associated with significant changes in CD3+, CD8+, or CD68+ immune cell composition (data not shown).
Tumor cell expression of PD-L1 was especially prominent in foci of tumor squamous metaplasia, foci of myometrial invasion, and foci of lymphatic/vascular space invasion (Figure 2 C, D). When present, squamous differentiation appeared to be more strongly associated with PD-L1 expression. Absence of squamous differentiation was noted in 74% of the PD-L1 negative cases, but only in 29% of the PD-L1 positive cases. A subset of tumors (21/63 PD-L1 positive cases, 33%; 21/132 total cases, 16%) had higher PD-L1 expression, characterized as either increased percentage of tumor cells positively staining or the presence of 3+ staining intensity. Sixteen of 63 cases (25%) had 2-5% of tumor staining, 3 cases (5%) had 6-20% of tumor staining, and 1 case (2%) had 75% of tumor staining. One case with 1% of tumor staining positive for PD-L1 was included in the PD-L1 high subset because it had stronger intensity of staining (3+) in tumor cells. Only 10% of the tumors in the PD-L1 high subset lacked squamous differentiation, and in 86% of the PD-L1 high tumors the squamous portion was positive for PD-L1. Presence of PD-L1 high was associated with significantly increased CD3+ and CD8+ lymphocytes in the tumor hotspot and significantly increased CD3+ lymphocytes in the tumor periphery compared to PD-L1 negative cases (Table 3). The number of CD68+ macrophages was not significantly different between PD-L1 high and PD-L1 negative cases (Table 3). Higher levels of PD-L1 were not significantly associated with any of the other clinical or pathological variables studied (Table  3). POLE mutation has been previously associated with increased tumor infiltrating lymphocytes (4). We hypothesized that because of its association with higher CD3+ and CD8+ lymphocytes, the PD-L1 high subset would be enriched with tumors with POLE mutation. However, only 1 mutation, a POLE exon 9 mutation, was identified in the PD-L1 high subset. This case was from a 60 year-old patient with a stage I, grade 2 endometrioid carcinoma with less than 50% myometrial invasion. Microscopically, this tumor did not have distinct features. Lymphatic/vascular space invasion was absent, and there was no metastasis in 12 surgically resected lymph nodes or the omentum. The tumor had 3+ strength of tumor staining for PD-L1 in approximately 3% of the tumor cells. The number of CD3+, CD8+, and CD68+ cells were not significantly higher in the one POLE mutant tumor compared to the POLE wildtype tumors (data not shown).
Higher expression of PD-L1 has been observed in grade 3 endometrioid and nonendometrioid carcinomas (10). As these histotypes are associated with TP53 gene mutation and/or immunohistochemistry patterns suggestive of TP53 gene mutation, we speculated that the PD-L1 high subset was enriched with tumors with TP53 gene mutation. However, all of the 21 tumors in this subset had scattered, weak nuclear expression of p53, associated with TP53 gene wildtype (data not shown). Seven of the 21 tumors in the PD-L1 high subset had nuclear localization of β-catenin, associated with CTNNB1 gene mutation. The CD3+, CD8+, and CD68+ immune cell infiltrates in these 7 tumors were not significantly different compared to the 14 PD-L1 high cases without nuclear β-catenin (data not shown).
PTEN loss is one of the most common molecular events in endometrioid-type endometrial carcinoma (12). In the 132 carcinomas examined in this study, 45% had PTEN protein loss by immunohistochemistry (Table 1). In melanoma, PTEN loss is associated with fewer CD8+ lymphocytes at the tumor site, decreased T-lymphocyte-mediated cell death, and decreased response to PD-1 blockade (19). In the current study, PTEN loss was not significantly associated with expression of PD-L1 or PD-L1 high (Tables 2 and 3). Independent of tumor PD-L1 status, there were no significant differences in numbers of CD3+ lymphocytes, CD8+ lymphocytes, or CD68+ macrophages in PTEN positive vs. PTEN negative tumors (Table 4).

Discussion
Efforts to identify biomarkers associated with endometrial cancer patients most likely to respond to immune based therapeutic approaches are important. Endometrial cancer is the most common gynecologic malignancy, with an estimated 63,230 new diagnoses in 2018 (20). In contrast to many other common cancer types, incidence and annual deaths from endometrial cancer are increasing (20,21). There are few therapeutic options for women with advanced or recurrent endometrial cancer, and no new agents have been approved in the past few decades (22,23). Therefore, immune-based approaches, in carefully chosen patients, have the potential to make a real impact in this disease.
In this study, we identified a subset (16%) of microsatellite-stable, mismatch repair intact endometrioid type endometrial carcinomas with increased tumor PD-L1 immunohistochemical staining (PD-L1 high positive subgroup). The tumors with increased tumor PD-L1 expression also showed increased numbers of CD3+ and CD8+ lymphocytes. This PD-L1 high group was not enriched with POLE mutations. These findings support the idea proposed by Shukla et al. (9) that there is a subset of POLE wildtype, mismatch repair intact endometrial cancers with increased tumor-associated lymphocytes. As yet unknown molecular mechanisms may underlie the recruitment of immune cells to these particular endometrioid carcinomas. Data from immunotherapy clinical trials will be necessary to determine if this subset is also more responsive to this therapeutic approach.
Although PD-L1 expression has been shown to be more frequent in microsatellite instability-high/POLE-mutated endometrial cancer (4-6, 10), PD-L1 staining has also been reported in microsatellite-stable endometrial carcinoma. In recent studies, the reported percentage of mismatch repair intact endometrial carcinomas with tumor cell expression of PD-L1 ranged from 4-20% (4, 5, 10); this range is comparable to the percentage of PD-L1 high (16%) in the current study. The variable percentage of cases positive for PD-L1 may be due to varying methodologies, including different PD-L1 antibodies, inclusion of endometrioid versus non-endometrioid histologies, stochastic geographic differences in PD-L1 expression, as well as tissue sampling by microarray versus full tissue sections. To keep tumor histology constant, the current study only included patients with FIGO grade 2 endometrioid endometrial carcinomas. It has been previously shown that lower grade endometrioid tumors have lower PD-L1 expression compared to grade 3 endometrioid carcinomas and non-endometrioid carcinomas (10). The detection of any tumoral staining was higher in the current study (48%) as compared to these previous studies. This may be due, in part, to the fact that full tissue sections of tumor were used in the current study, as the staining by PD-L1 can be very focal (1% tumor cells positive in the majority of cases in this study). Given that we frequently observed positive PD-L1 tumor staining in the invasive front, inclusion of the periphery/invasive front should be considered if tissue microarray analyses are performed. Overall, the focal and weak PD-L1 staining in the majority of cases of mismatch repair intact endometrial cancers in our study is consistent with the results of previous published work.
The immunohistochemical expression characteristics of PD-L1 identified in this study provide useful insight into the staining pattern in endometrial cancer. Cancers with metaplastic changes, such as squamous metaplasia, were noted to have increased PD-L1 staining ( Figure 2). Tumor PD-L1 positivity was frequently noted in the invasive front in the tumor PD-L1 high subgroup. In addition, staining was noted more frequently in cases with lymphatic/vascular space invasion, which was also previously reported (10). This raises the possibility that the microcystic elongated fragmented pattern of endometrial cancer invasion (24) could be associated with PD-L1 expression, due to its morphologic associations with metaplastic changes and lymphatic/vascular space invasion. The current study was not optimally designed to examine any possible association between PD-L1 and this distinctive invasive pattern, as only 5% of the 132 endometrial cancers examined showed this invasion pattern. This relatively low percentage is likely due to the fact that 99/132 (75%) of the cases included in this study had no myometrial invasion or less than 50% myometrial thickness invasion (stage IA). The PD-L1 high subset was not associated with aberrant p53 tumor expression. Nuclear translocation of β-catenin did not preferentially occur in the PD-L1 high subset. Thus, other than the presence of squamous metaplasia, which is very common in endoometrioid carcinomas, and increased numbers of CD3 positive and CD8 positive lymphocytes, this PD-L1 high subset does not have distinctive clinical or pathological features. Based on these results, this subset would reside in the copy number-low TCGA group of endometrial cancers, which is the largest group (1).
Tumors with POLE mutation, high levels of microsatellite instability, or high tumor mutation burden all seem to have in common a higher tumor associated immune cell infiltrate. This is important, as evidence suggests that increased tumor associated immune cells is associated with better survival and improved responses to checkpoint inhibitor therapy. In a mouse model of melanoma, pre-treatment with a PI3Kβ inhibitor resulted in increased tumor T lymphocytes and improved responses to anti-CTLA-4 and anti-PD-1 therapy (19). Responders to PD-1 blockade have significantly increased density in CD3+, CD8+, and CD45RO+ T lymphocytes in melanoma (25). In colorectal adenocarcinoma, higher intra-tumoral CD8+ T cells are associated with objective response to pembrolizumab (7). Higher expression in the tumor of CD8 mRNA is associated with higher PD-L1 mRNA expression and significantly increased survival in endometrioid-type endometrial carcinoma (26). It has been argued that both increased tumor associated lymphocytes and increased PD-L1 expression on tumor cells are necessary for optimal response to checkpoint inhibitor blockade (13).
An emerging area of study is the concept that tumor associated mutations may act as molecular modifiers of immune cell function. PTEN loss has been shown to be associated with decreased CD8+ immune cells and resistance to anti-PD-1 therapy in melanoma (19). PTEN loss does not seem to be associated with increased PD-L1 expression in melanoma (19). It was proposed that PTEN loss and PD-L1 expression were occurring due to two separate mechanisms that evolved independently to influence the immune microenvironment. An association with PTEN loss and PD-L1 expression has been reported in other tumor types such as glioblastoma multiforme and colorectal cancer (27). In the current study, PTEN loss had no statistically significant impact on PD-L1 expression or the density of CD3, CD8, and CD68 tumor associated immune cells. This suggests that molecular modifiers of the local tumor immune response may be tumor type-specific. Using a pan-cancer analytic approach, tumor driver mutations in CTNNB1, NRAS, and IDH1 have been associated with lower levels of immune cells in tumors (28). NRAS and IDH1 mutations are uncommon in endometrial cancer, but CTNNB1 mutation occurs in approximately 20% of endometrioid endometrial carcinomas and has been associated with decreased recurrence-free survival (29,30). Activation of the Wnt/β-catenin pathway in melanoma is associated with local immune suppression (31). Such a mechanism of local immune suppression by CTNNB1 mutation or activation of the Wnt/β-catenin pathway has not yet been described for endometrial cancer, but it could explain, at least in part, the worse survival in this group of patients. Schematic overview of study design. A total of 382 endometrial cancers were tested for mismatch repair immunohistochemistry and/or PCR-based microsatellite instability analysis. From this group, 132 FIGO grade 2, microsatellite-stable endometrial cancers with intact expression of mismatch repair proteins were identified. PD-L1, PTEN, CD3, CD8, and CD68 immunohistochemical analyses were performed in this set of tumors.


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Critical illness and the role of the microbiome

The number of microbes living within the intestinal lumen is similar to the number of all cells of human origin in the host. Although historically little attention has been paid to the massive microbial community residing inside each of us, the last few years have witnessed an explosion of information related to the role of the microbiome in the maintenance of health and in the pathogenesis of disease. Here, we review data suggesting that the microbiome is converted into a pathobiome in critical illness and potential strategies for targeting the microbiome for therapeutic gain in the intensive care unit.

INTRODUCTION
A pproximately 40 trillion microorganisms reside inside the intestine. 1 Under basal conditions, the microbiome is not simply an innocent bystander, living peacefully side-by-side with its human host. Rather, commensal microbes are health-promoting and play numerous diverse roles in the maintenance of human wellness. In contrast, the microbiome is severely altered in multiple disease states with the conversion of the health-inducing microbiome into a disease-promoting microbiome (also known as a pathobiome). 2 These perturbations are particularly pronounced in the intensive care unit (ICU), where the gut has long been hypothesized to be "the motor" of critical illness. 3,4 The largest study to date on the microbiome in critical illness compared fecal samples from 115 adult ICU patients in four medical centers within 48 h of admission and at discharge or on ICU day 10 to 1,242 healthy patients. 5 Critically ill patients had rapid depletion of health-promoting organisms and overgrowth of known pathogens. Specifically, when examining phylum-level taxonomy, the common gram-positive Firmicutes and Gram-negative Bacteroidetes were both decreased, as was Faecalibacterium, an antiinflammatory organism. In contrast, potential pathogens such as Enterobacter and Staphylococcus were increased, and there was a relative increase in Proteobacteria. Phylogentic diversity was also significantly decreased at discharge compared to admission. Of note, this study used 16S rRNA gene amplicon sequencing, which does not provide sufficient genomic resolution to identify bacterial species, but identifies genera instead. This is important because genera cannot distinguish between pathogenic and non-pathogenic species (Staphylococcus aureus versus other non-pathogenic Staphylococci, for instance).
Similarly, a prospective observational study of 34 ICU patients (both septic and non-septic) and 15 healthy controls showed a marked shift in fecal bacterial composition in critically ill patients with disappearance of bacteria genera with key functions in host metabolism. 6 Notably, there were extreme individual differences in 13 critically ill patients with a single bacterial genus making up more than 50% of the gut microbiota. However, no association was identified between microbial diversity, Firmicutes/Bacteroidetes ratio or Gram-positive/Gram-negative ratio and survival or complications. This remarkable loss of diversity mimics a study of 14 ICU patients that reported the emergence of ultra-lowdiversity communities in 35% of patients containing only one to four bacterial taxa. 7 Similar to the above studies, at the phylum level, the communities most commonly contained Enterococcus, Staphylococcus, and Enterobacter. Notably, cultured stool samples correlated to the 16S rRNA analysis, but also revealed the emergence of Candida albicans and Candida glabrata in 75% of patients.
It is crucial to note that microbiome composition is not static but rapidly evolves over time in the ICU and with severity of illness. It was initially reported nearly 50 years ago that the prevalence of Gram-negative oropharyngeal bacteria is low in physiologically normal subjects despite hospital exposure but increases markedly in sick patients, and this increased prevalence correlates most closely with severity of illness. 8 More recently, a pilot study of 12 adult ICU patients examined stool samples on mechanically ventilated patients on days 1-2, 2-4, 5-8, and 7-10. 9 Bacteria belonging to the phyla Firmicutes and Bacteroidetes were predominant in all samples, but the percentages changed markedly over time. Notably, a Bacteroidetes/Firmicutes ratio of >10 was seen in four of six non-survivors, whereas a ratio of <0.1 was seen in one non-survivor. No survivor had a ratio of >10 or <0.1, although this small study was underpowered to draw any conclusions related to the relationship between this ratio and mortality.
The microbiome is also altered in critically ill children. A comparison of 37 pediatric ICU patients with a mean age of 2.9 years to both pediatric and adult reference datasets demonstrated that pediatric ICU patients had decreased diversity with enrichment at the genus level of Enterococcus and Staphylococcus at multiple body sites with depletion of commensals such as Faecalibacterium and Ruminococcus from the gut. 10 Notably, both alpha and beta diversity were unstable over time in patients followed longitudinally.
The microbiome is not restricted to the gut and multiple body sites contain microbes that have been implicated in critical illness. For instance, a study comparing 15 patients requiring mechanical ventilation to healthy subjects who had lower respiratory tract sampling by bronchoscopy showed that both upper and lower respiratory tract microbiota diversity were decreased within 24 h of intubation and further decreased over time. 11 Furthermore, in a study of patients admitted to the ICU after severe blunt trauma, smoking prior to the ICU admission was significantly associated with microbial composition both at ICU admission and at 48 h, and this also was associated with development of acute respiratory distress syndrome. 12 The lung microbiota was also reported to be altered in a mouse study of acute lung injury induced by lipopolysaccharide. 13 In addition, emerging evidence suggests that gut-derived bacteria travel to other body sites in critical illness. Using culture-independent evidence in a murine model of sepsis, lung communities were dominated by gut-associated bacteria, and ecological analysis revealed the lower gastrointestinal tract as the likely source of post-sepsis lung bacteria (rather than the upper respiratory tract). In addition, gut-specific bacteria were abundant in patients with acute respiratory distress syndrome. 14 Similarly, gut-associated bacteria were enriched in the brains of mice 5 days after abdominal sepsis, and this was associated with severity of neuroinflammation. 15 The etiology behind the changes to the microbiome in critical illness is almost assuredly multifactorial. Plausible causes include changes induced by the critically ill state as well as unintentional side-effects of treatments of critical illness (e.g. antimicrobial therapy, opiates, proton pump inhibitors, and tube feeding). It is often difficult to uncouple the effects of critical illness from the impact of antibiotics on the microbiome in the ICU, as the majority of ICU patients receive antimicrobial therapy at some point during their hospitalization. Furthermore, as the microbiome acts as a major modulator of innate immunity, it is theoretically possible that antibiotics alter immune responsiveness by altering the microbiome (distinct from their intended antimicrobial action). In an attempt to model this, a proof-of-principle trial randomized 16 healthy men to receive either broad spectrum antibiotics or no treatment for 7 days followed by a single dose of endotoxin, designed to mimic a transient septic-like state. 16 As expected, microbial diversity was significantly diminished by the treatment with antibiotics. Following endotoxemia, however, no differences were noted in neutrophil influx, cytokine production, coagulation activation, endothelial activation, or leukocyte responsiveness to multiple Toll-like receptor ligands or clinically relevant bacteria ex vivo. This study is reassuring on some levels; however, the relevance of these findings to septic patients with ongoing infection, antimicrobial therapy, physiologic perturbance, and organ failure is not clear. It should be noted, however, that a study of 15 critically ill patients without antibiotic exposure reported significant changes in their microbiome within 6 h of their arrival at the emergency room compared to healthy volunteers. 17 Unfortunately, the concept of "good" and "bad" bacteria is likely overly simplistic as bacteria can alter their own virulence depending on host factors, so the identical bacterial species can be adaptive or maladaptive depending on the clinical situation. Under basal conditions, bacteria rarely express virulence genes. However, in settings of host stress when resources are limited, bacteria can develop both ancestral and newly acquired resistance genes. This could lead to bacterial invasion and, in turn, drive a maladaptive host response. 2 Notably, the timescale in which bacteria can shift their evolutionary trajectories is much shorter (hours) than that of the human host (days to weeks). Thus, the inner microbial world within us has the capacity to adapt to changes faster than the critically ill patient does, which can potentially be devastating if the microbial response is to aggressively attack its host. An elegant preclinical example of the implications of this was published by Alverdy's group. 18 Both healthy mice and mice that underwent a non-lethal 30% hepatectomy were injected with Pseudomonas aeruginosa into the cecum. The bacteria were then withdrawn and injected into the peritoneum of uninjured mice. Animals that received bacteria from healthy mice all survived, yet all animals that received bacteria from mice that had a 30% hepatectomy died. The underlying mechanism is that bacteria injected into mice with a hepatectomy sensed host stress and, in turn, induced virulence factors that subsequently killed the uninjured mouse. As identical bacteria were used in this experiment, this highlights the importance of the host environment in impacting the microbial community, which, in turn, directly impacts the health of the host.
Numerous therapeutic strategies currently exist for manipulating the microbiome in the ICU. These include probiotics, fecal microbial transplant (FMT), and selective decontamination of the digestive tract (SDD). Each of these has shown some promise, yet each also has significant challenges both logistically and intellectually. Probiotics are selective exogenous bacteria given to the host. Meta-analyses and multiple studies have indicated that probiotics are effective at decreasing ventilator-associated pneumonia 19-21 but do not alter length of stay or mortality. A clear limitation to the published works on probiotics is the significant heterogeneity between studies in dose, length, and bacteria given. In addition, most of the studies on probiotics were undertaken prior to our current understanding of the microbiome, implying a more mechanistic design in the future might have greater efficacy. Related strategies are prebiotics, which are non-digestible nutrients that stimulate commensal bacterial growth, and synbiotics (a combination of probiotics and prebiotics). Prebiotics directly regulate host mucosal signaling to alter the response to bacterial infection; 22 however, clinical data are still preliminary. 23 Instead of giving selective bacteria, FMT transfers an entire microbiome from a healthy patient to a diseased one, with the goal of restoring a normal microenvironment. Fecal microbial transplant has proven to be remarkably successful in recurrent Clostridia difficile colitis, where a recent meta-analysis of 37 trials shows 92% resolution and a relative risk of 0.23 compared to oral vancomycin, 24 similar to the original landmark study of FMT showing a 93.8% cure rate. 25 However, the usage of FMT in the ICU is currently limited to a small number of case reports. 26 Most ICU patients receive antibiotics that would be expected to immediately alter the transplanted microbial community. In addition, the long-term impact of giving FMT in the ICU is unknown.
Selective decontamination of the digestive tract represents an opposite approach to probiotics or FMT. Rather than augmenting healthy bacteria or stimulating bacterial growth, SDD seeks to decrease pathogenic bacteria. Selective decontamination of the digestive tract is somewhat of a misnomer as patients receive systemic antibiotics in addition to topical antibiotics. Regardless, this approach has been shown to be very effective with a meta-analysis of nearly 30 high quality trials showing a reduction in mortality with a relative risk of 0.73. 27 However, each of the source studies was undertaken in countries with low basal antimicrobial resistance. Although there is minimal real-world evidence that SDD induces antimicrobial resistance, its use is currently limited to a few countries because of the theoretical concern that SDD could induce resistance.
Conceptually, tricking bacteria into believing a host is healthy could prevent induction of virulence factors. Although this is not feasible at the bedside currently, preclinical work suggests this might be a viable strategy in the future. A key determinant of induction of bacterial virulence factors is lack of intraluminal phosphate. Phosphate-polyethylene glycol is a novel preclinical therapeutic that creates local phosphate abundance. When given to septic mice, it prevents development of virulence and importantly improves survival. 28 Although innumerable insights about the microbiome remain to be discovered, a rapidly evolving understanding of our inner microbial community and its interaction with the host suggests the microbiome could play a central role in the pathophysiology of critical illness. This is conceptually important as a recent review of 51 studies of animals and pediatric and adult ICU patients illustrates multiple ways in which the microbiome can be altered, including metabolomic and proteomic changes, alterations induced by nutrition, and alterations in antibiotic resistance genes. 29 Therapeutic strategies in the future will likely be targeted to restoration of the healthy microbiome, potentially combined with strategies to prevent the development of microbial virulence.


extfieldsofstudy: Chemistry Medicine

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Aryl Hydrocarbon Receptor Antagonists Mitigate the Effects of Dioxin on Critical Cellular Functions in Differentiating Human Osteoblast-Like Cells

The inhibition of bone healing in humans is a well-established effect associated with cigarette smoking, but the underlying mechanisms are still unclear. Recent work using animal cell lines have implicated the aryl hydrocarbon receptor (AhR) as a mediator of the anti-osteogenic effects of cigarette smoke, but the complexity of cigarette smoke mixtures makes understanding the mechanisms of action a major challenge. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a high-affinity AhR ligand that is frequently used to investigate biological processes impacted by AhR activation. Since there are dozens of AhR ligands present in cigarette smoke, we utilized dioxin as a prototype ligand to activate the receptor and explore its effects on pro-osteogenic biomarkers and other factors critical to osteogenesis using a human osteoblast-like cell line. We also explored the capacity for AhR antagonists to protect against dioxin action in this context. We found dioxin to inhibit osteogenic differentiation, whereas co-treatment with various AhR antagonists protected against dioxin action. Dioxin also negatively impacted cell adhesion with a corresponding reduction in the expression of integrin and cadherin proteins, which are known to be involved in this process. Similarly, the dioxin-mediated inhibition of cell migration correlated with reduced expression of the chemokine receptor CXCR4 and its ligand, CXCL12, and co-treatment with antagonists restored migratory capacity. Our results suggest that AhR activation may play a role in the bone regenerative response in humans exposed to AhR activators, such as those present in cigarette smoke. Given the similarity of our results using a human cell line to previous work done in murine cells, animal models may yield data relevant to the human setting. In addition, the AhR may represent a potential therapeutic target for orthopedic patients who smoke cigarettes, or those who are exposed to secondhand smoke or other environmental sources of aryl hydrocarbons.

Introduction
The influence of tobacco smoke on human health remains an important problem worldwide. Cigarette smoke (CS) has a well-established role in the pathogenesis of numerous smoking-related disorders, including chronic obstructive pulmonary disease (COPD), lung cancer, and atherosclerosis [1,2]. However, smoking also advances musculoskeletal disease and significantly impacts the treatment of orthopedic conditions [3]. In addition to promoting osteoporosis, degenerative disc disease, and wound complications, smoking significantly inhibits osseointegration and bony union [4], which are associated with higher rates of revision procedures [5]. Smoking has also been shown to have a negative impact on outcomes after spine surgery, with a nonunion rate nearly double that of non-smokers (14.2% vs. 26.5%) [6].
Because CS contains more than 4000 distinct chemical constituents, determining a singular mechanism by which cigarette smoke inhibits bone growth is impossible [7]. Nicotine-which is present in the tar phase of cigarette smoke-has been suggested as a major mediator but its effects on osteoblasts and bone healing are nuanced; high concentrations of nicotine have been shown to inhibit osteoblast proliferation, whereas low concentrations may actually have a proliferative effect [8].
to prevent the adverse effects of cigarette smoke and other environmental sources of AhR activators on bone healing.

Dioxin Exposure and AhR Activation
At a dose of 100 nM, dioxin exposure did not change the overall cell number in standard or osteogenic conditions for the time periods evaluated in this study; this dose was chosen for subsequent differentiation experiments. Cells were analyzed for CYP1A1 expression as a marker of dioxin exposure and subsequent AhR activation. Under standard conditions, mRNA expression for the AhR-dependent CYP1A1 gene increased 2.36-fold after treatment with dioxin relative to dimethyl sulfoxide (DMSO)-treated cells (p < 0.05, Figure 1). Under osteogenic conditions, dioxin induced CYP1A1 mRNA 8.72-fold over vehicle control-treated cells.

Dioxin Exposure and AhR Activation
At a dose of 100 nM, dioxin exposure did not change the overall cell number in standard or osteogenic conditions for the time periods evaluated in this study; this dose was chosen for subsequent differentiation experiments. Cells were analyzed for CYP1A1 expression as a marker of dioxin exposure and subsequent AhR activation. Under standard conditions, mRNA expression for the AhR-dependent CYP1A1 gene increased 2.36-fold after treatment with dioxin relative to dimethyl sulfoxide (DMSO)-treated cells (p < 0.05, Figure 1). Under osteogenic conditions, dioxin induced CYP1A1 mRNA 8.72-fold over vehicle control-treated cells.

Early Markers of Osteogenic Differentiation
As expected, RUNX2 expression in MG-63 cells was upregulated under osteogenic conditions relative to standard conditions. The mRNA was decreased by 0.79-fold and the protein level was decreased by 1.55-fold (Figure 2A,B) in cells treated with 100 nM dioxin relative to control-treated cells (p < 0.05). Alkaline phosphatase (ALP) gene expression was significantly increased under osteogenic conditions relative to standard conditions, but dioxin treatment inhibited the induction of ALP mRNA relative to DMSO-treated control cells (6.0-vs. 3.5-fold induction over standard media DMSO control; p < 0.05; Figure 2C). Similarly, ALP enzymatic activity was significantly increased in osteogenic media (OM) relative to standard media, as expected (p < 0.05, Figure 2D). Dioxin significantly inhibited osteogenic media-induced ALP activity at all doses tested. This effect was dose-dependent, with a significant reduction from 10 nM dioxin.

Cell Adhesion
Cell adhesion rates were quantified using a modified MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay. MG-63 cells grown in the presence of dioxin under both standard ( Figure 3A) and osteogenic ( Figure 3B) conditions showed significantly reduced adhesion at nearly all time points relative to vehicle control-treated cells (p < 0.05). Cell adhesion was also visualized and quantified by measuring average cell diameter after rhodamine-conjugated phalloidin staining ( Figure 3C). The average diameter of the cells was significantly lower in dioxin-treated cultures grown in both standard (72 µm vs. 41 µm) and osteogenic media conditions (38 µm vs. 32 µm), relative to respective controls (p < 0.05). Dioxin induces CYP1A1 under both standard and osteogenic conditions. Expression of CYP1A1 mRNA after treatment with 100 nM dioxin or dimethyl sulfoxide (DMSO) vehicle control in cells grown under standard growth medium or osteogenic induction medium confirms that the AhR pathway is intact and functional in MG-63 cells. mRNA expression levels were normalized to DMSO vehicle control-treated cells grown in standard media. * p < 0.05 relative to 0 nM dioxin under standard or osteogenic conditions. Error bar means ± SEM.

Early Markers of Osteogenic Differentiation
As expected, RUNX2 expression in MG-63 cells was upregulated under osteogenic conditions relative to standard conditions. The mRNA was decreased by 0.79-fold and the protein level was decreased by 1.55-fold (Figure 2A,B) in cells treated with 100 nM dioxin relative to control-treated cells (p < 0.05). Alkaline phosphatase (ALP) gene expression was significantly increased under osteogenic conditions relative to standard conditions, but dioxin treatment inhibited the induction of ALP mRNA relative to DMSO-treated control cells (6.0-vs. 3.5-fold induction over standard media DMSO control; p < 0.05; Figure 2C). Similarly, ALP enzymatic activity was significantly increased in osteogenic media (OM) relative to standard media, as expected (p < 0.05, Figure 2D). Dioxin significantly inhibited osteogenic media-induced ALP activity at all doses tested. This effect was dose-dependent, with a significant reduction from 10 nM dioxin.

Cell Adhesion
Cell adhesion rates were quantified using a modified MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay. MG-63 cells grown in the presence of dioxin under both standard ( Figure 3A) and osteogenic ( Figure 3B) conditions showed significantly reduced adhesion at nearly all time points relative to vehicle control-treated cells (p < 0.05). Cell adhesion was also visualized and quantified by measuring average cell diameter after rhodamine-conjugated phalloidin staining ( Figure 3C). The average diameter of the cells was significantly lower in dioxin-treated cultures grown in both standard (72 µm vs. 41 µm) and osteogenic media conditions (38 µm vs. 32 µm), relative to respective controls (p < 0.05). Integrins and cadherins have been shown to play an important role in the control of osteogenesis and osteogenic differentiation [24]. Indeed, we found that dioxin exposure affected the expression of integrin and cadherin proteins that have important functions in cell-extracellular matrix interactions (p < 0.05, Figure 3D). Dioxin significantly downregulated expression of integrin α5 under both standard and osteogenic conditions (1.00 vs. 0.45 and 1.0 vs. 0.22 relative expression levels, respectively) and E-cadherin protein expression (1.00 vs. 0.74 and 1.00 vs. 0.23, respectively), whereas integrin αV and integrin β1 levels were unchanged. Interestingly, N-cadherin expression was decreased by 77% in dioxin-treated cells cultured under osteogenic conditions (1.00 vs. 0.23 relative expression levels).

Cell Migration
The effect of dioxin exposure on the migratory capacity of MG-63 cells was assessed via wound healing and transwell chamber migration assays. Dioxin-treated cells showed a reduced capacity for migration across the "wound" space relative to DMSO-treated cells after 15 h (82% vs. 65%, respectively; p < 0.05, Figure 4A). In directional migration assays, the presence of FBS in the lower chamber significantly induced migration in control-treated cells as expected. However, dioxin pretreatment caused a significant dose-dependent reduction in directional migratory capacity (at 20 nM and 100 nM dioxin) relative to control-treated cells (100% vs. 30% and 100% vs. 15%, respectively; p < 0.05, Figure 4B).
CXCL12 (SDF-1) and its receptor, CXCR4, are known to play an important role in bone formation by influencing BMP-2 signaling [25]. Interestingly, CXCL12 levels are increased at the site of injury Integrins and cadherins have been shown to play an important role in the control of osteogenesis and osteogenic differentiation [24]. Indeed, we found that dioxin exposure affected the expression of integrin and cadherin proteins that have important functions in cell-extracellular matrix interactions (p < 0.05, Figure 3D). Dioxin significantly downregulated expression of integrin α5 under both standard and osteogenic conditions (1.00 vs. 0.45 and 1.0 vs. 0.22 relative expression levels, respectively) and E-cadherin protein expression (1.00 vs. 0.74 and 1.00 vs. 0.23, respectively), whereas integrin αV and integrin β1 levels were unchanged. Interestingly, N-cadherin expression was decreased by 77% in dioxin-treated cells cultured under osteogenic conditions (1.00 vs. 0.23 relative expression levels).

Cell Migration
The effect of dioxin exposure on the migratory capacity of MG-63 cells was assessed via wound healing and transwell chamber migration assays. Dioxin-treated cells showed a reduced capacity for migration across the "wound" space relative to DMSO-treated cells after 15 h (82% vs. 65%, respectively; p < 0.05, Figure 4A). In directional migration assays, the presence of FBS in the lower chamber significantly induced migration in control-treated cells as expected. However, dioxin pre-treatment caused a significant dose-dependent reduction in directional migratory capacity (at 20 nM and 100 nM dioxin) relative to control-treated cells (100% vs. 30% and 100% vs. 15%, respectively; p < 0.05, Figure 4B).
CXCL12 (SDF-1) and its receptor, CXCR4, are known to play an important role in bone formation by influencing BMP-2 signaling [25]. Interestingly, CXCL12 levels are increased at the site of injury and are thought to mediate the honing of progenitor cells to sites of bony injury [26,27]. We hypothesized that dioxin-treated cells, which have reduced migratory capacity, may express reduced levels of CXCL12 and CXCR4 relative to DMSO-treated controls. Indeed, mRNA expression of CXCL12 was decreased in dioxin-treated cells (1.00 vs. 0.42 relative expression levels). Similarly, both mRNA and protein expression of CXCR4 were decreased after dioxin exposure (1.00 vs. 0.64 and 1.00 vs. 0.03 relative expression levels, respectively; p < 0.05, Figure 4C).  Since mitogen-activated protein kinases (MAPKs) play a crucial role in cell migration [28], we also quantified the expression levels of total and active p38 and ERK1/2. Dioxin treatment significantly decreased the expression of total and active (p-p38) p38 (1.00 vs. 0.25 and 1.00 vs. 0.03, respectively), as well as total and active (p-ERK1/2) ERK1/2 (1.00 vs. 0.42 and 1.00 vs. 0.05, respectively; p < 0.05, Figure 4D). and E-cadherin protein expression levels were significantly decreased in dioxin-exposed cells under both standard and osteogenic conditions, whereas INTαV and INTβ1 were unchanged. N-cadherin levels were decreased only in differentiating dioxin-treated cells. * p < 0.05 relative to 0 nM dioxin under standard or osteogenic conditions. Error bar means ± SEM.
Since mitogen-activated protein kinases (MAPKs) play a crucial role in cell migration [28], we also quantified the expression levels of total and active p38 and ERK1/2. Dioxin treatment significantly decreased the expression of total and active (p-p38) p38 (1.00 vs. 0.25 and 1.00 vs. 0.03, respectively), as well as total and active (p-ERK1/2) ERK1/2 (1.00 vs. 0.42 and 1.00 vs. 0.05, respectively; p < 0.05, Figure 4D). Rapid actin polymerization is important for cell motility and is necessary to push forward the leading edge of migrating cells [29]. We, therefore, hypothesized that reduced cell migration after dioxin exposure may be in part due to a decrease in the rate of actin polymerization. To test this hypothesis, cells were pre-treated with DMSO or dioxin for 3 days and followed by treatment with actin de-polymerization reagent, cytochalasin D (cyto D), for 60 min. After release from cyto D for 40 min, both DMSO-and dioxin-treated cells were alyzed using Rhodamine-conjugated phalloidin.
Cells showed visible re-polymerization. However, cells pre-treated with dioxin qualitatively showed reduced organization of actin filaments ( Figure 4E).

Matrix Mineralization
Matrix mineralization by MG-63 cells was visualized using both alizarin red and von Kossa staining. Cells were treated with increasing concentrations of dioxin under osteogenic conditions. For vehicle control-treated cells, the introduction of an osteogenic environment resulted in significantly elevated mineralization relative to cells grown in standard media as expected as determined by alizarin red staining ( Figure 5A).
Increasing concentrations of dioxin under osteogenic conditions dose-dependently corresponded with decreasing levels of mineral deposition with significance at all concentrations The presence of FBS in the lower chamber significantly increased the migration rate of DMSO-treated cells, whereas dioxin dose-dependently inhibited cell migration. Representative images of cells following migration towards an FBS gradient are shown; (C) mRNA expression levels of both CXCL12 and its receptor, CXCR4, were decreased in dioxin-treated cells, and CXCR4 protein expression was also decreased following dioxin exposure; (D) Dioxin significantly decreased the protein expression of total p38, active (phosphorylated) p38 (p-p38), total ERK1/2, and active (phosphorylated) ERK1/2 (p-ERK1/2). * p < 0.05 relative to 0 nM dioxin under standard or osteogenic conditions (A-D); (E) Dioxin inhibits cell adhesion/spreading, which was visualized by actin filament staining 40 min after release from cytochalasin D (cyto D) treatment. Rhodamine-bound F-actin is shown in red whereas nuclei are shown blue. Error bar means ± SEM. Rapid actin polymerization is important for cell motility and is necessary to push forward the leading edge of migrating cells [29]. We, therefore, hypothesized that reduced cell migration after dioxin exposure may be in part due to a decrease in the rate of actin polymerization. To test this hypothesis, cells were pre-treated with DMSO or dioxin for 3 days and followed by treatment with actin de-polymerization reagent, cytochalasin D (cyto D), for 60 min. After release from cyto D for 40 min, both DMSO-and dioxin-treated cells were alyzed using Rhodamine-conjugated phalloidin.
Cells showed visible re-polymerization. However, cells pre-treated with dioxin qualitatively showed reduced organization of actin filaments ( Figure 4E).

Matrix Mineralization
Matrix mineralization by MG-63 cells was visualized using both alizarin red and von Kossa staining. Cells were treated with increasing concentrations of dioxin under osteogenic conditions. For vehicle control-treated cells, the introduction of an osteogenic environment resulted in significantly elevated mineralization relative to cells grown in standard media as expected as determined by alizarin red staining ( Figure 5A).
Increasing concentrations of dioxin under osteogenic conditions dose-dependently corresponded with decreasing levels of mineral deposition with significance at all concentrations tested relative to vehicle-treated cells (p < 0.05, Figure 5B). The effects of dioxin on matrix mineralization were further confirmed by von Kossa staining ( Figure 5C).
Factors known to influence mineralization were also impacted by dioxin exposure. Osteocalcin (OCN) is an osteoblast-specific marker, the expression of which has been demonstrated by staining in three-dimensional bone nodules derived from hESCs [30]. In our study, we found that dioxin decreased OCN mRNA both in standard and osteogenic media (1.00 vs. 0.4 and 0.78 vs. 0.38, respectively; p < 0.05, Figure 5D). PHEX is a gene that encodes for a protease responsible for the degradation and clearance of osteoblast mineralization-inhibiting acidic serine-aspartate-rich motif (ASARM) peptides as demonstrated both in vitro and in vivo [31]. tested relative to vehicle-treated cells (p < 0.05, Figure 5B). The effects of dioxin on matrix mineralization were further confirmed by von Kossa staining ( Figure 5C). Factors known to influence mineralization were also impacted by dioxin exposure. Osteocalcin (OCN) is an osteoblast-specific marker, the expression of which has been demonstrated by staining in three-dimensional bone nodules derived from hESCs [30]. In our study, we found that dioxin decreased OCN mRNA both in standard and osteogenic media (1.00 vs. 0.4 and 0.78 vs. 0.38, respectively; p < 0.05, Figure 5D). PHEX is a gene that encodes for a protease responsible for the degradation and clearance of osteoblast mineralization-inhibiting acidic serine-aspartate-rich motif (ASARM) peptides as demonstrated both in vitro and in vivo [31].

AhR Antagonist Studies
Since the effects of dioxin on bone formation are likely mediated at least in part by the AhR [12], we hypothesized that co-treatment with dioxin and the AhR antagonists ANF, resveratrol, or DIM could prevent the dioxin-mediated inhibition of osteogenic differentiation. In preliminary work, we showed that at the relevant time points, cell proliferation was not inhibited by 0.5 µM ANF, 4 µM

AhR Antagonist Studies
Since the effects of dioxin on bone formation are likely mediated at least in part by the AhR [12], we hypothesized that co-treatment with dioxin and the AhR antagonists ANF, resveratrol, or DIM could prevent the dioxin-mediated inhibition of osteogenic differentiation. In preliminary work, we showed that at the relevant time points, cell proliferation was not inhibited by 0.5 µM ANF, 4 µM Res, and 10 µM DIM, and we subsequently used these doses for co-treatment studies. Notably, CYP1A1 protein was increased in dioxin-treated samples, and this induction was significantly decreased by co-treatment with AhR antagonists to near or below baseline levels (p < 0.05, Figure 6A). Co-treatment with ANF, Res, and DIM all significantly increased CXCL12 mRNA. However, whereas ANF co-treatment returned CXCR4 mRNA to normal levels (vehicle control-treated), resveratrol and DIM and co-treatment increased CXCR4 mRNA to levels above the vehicle controls. (Figure 6B). Upon co-treatment with AhR antagonists, ALP levels were slightly increased over dioxin-treated samples but not fully recovered with antagonist co-treatment, whereas RUNX2 levels were elevated relative to DMSO-treated cells. CYP1A1 protein was increased in dioxin-treated samples, and this induction was significantly decreased by co-treatment with AhR antagonists to near or below baseline levels (p < 0.05, Figure 6A). Co-treatment with ANF, Res, and DIM all significantly increased CXCL12 mRNA. However, whereas ANF co-treatment returned CXCR4 mRNA to normal levels (vehicle control-treated), resveratrol and DIM and co-treatment increased CXCR4 mRNA to levels above the vehicle controls. (Figure 6B). Upon co-treatment with AhR antagonists, ALP levels were slightly increased over dioxin-treated samples but not fully recovered with antagonist co-treatment, whereas RUNX2 levels were elevated relative to DMSO-treated cells. Figure 6. Osteogenesis-related gene expression after dioxin and AhR antagonist co-treatment. (A) CYP1A1 protein levels returned to near-baseline levels with AhR antagonist co-treatment. * p < 0.05 relative to 100 nM dioxin condition; (B) CXCL12 mRNA expression was recovered by co-treatment with AhR antagonists. CXCR4 mRNA expression was restored to control levels or higher by cotreatment with resveratrol and 3,3′-diindolylmethane (DIM). ALP and RUNX2 mRNA expression was recovered by AhR antagonist co-treatment. * p < 0.05 relative to 100 nM dioxin condition, ^ p < 0.05 relative to 0 nM dioxin condition. Error bar means ± SEM.
Because dioxin inhibited the cell migration capacity (as shown in Figure 4), we investigated whether the antagonists could reduce the negative impact of dioxin on migration. In wound healing assays, co-treatment with AhR antagonists significantly increased cell migration across the wound space compared to MG-63 cells treated with dioxin alone (p < 0.05, Figure 7A). A similar rescue effect was recapitulated in ALP activity and mineralization assays, where each of the three antagonists tested provided a significant protective effect relative to dioxin alone (p < 0.05, Figure 7B,C). ANF demonstrated the strongest rescue effect on both cell migration and mineralization in dioxin-treated cells, with a full restoration to control levels; resveratrol and DIM also showed significant mitigating effects relative to dioxin-treated cells. (A) CYP1A1 protein levels returned to near-baseline levels with AhR antagonist co-treatment. * p < 0.05 relative to 100 nM dioxin condition; (B) CXCL12 mRNA expression was recovered by co-treatment with AhR antagonists. CXCR4 mRNA expression was restored to control levels or higher by co-treatment with resveratrol and 3,3 -diindolylmethane (DIM). ALP and RUNX2 mRNA expression was recovered by AhR antagonist co-treatment. * p < 0.05 relative to 100 nM dioxin condition,ˆp < 0.05 relative to 0 nM dioxin condition. Error bar means ± SEM.
Because dioxin inhibited the cell migration capacity (as shown in Figure 4), we investigated whether the antagonists could reduce the negative impact of dioxin on migration. In wound healing assays, co-treatment with AhR antagonists significantly increased cell migration across the wound space compared to MG-63 cells treated with dioxin alone (p < 0.05, Figure 7A). A similar rescue effect was recapitulated in ALP activity and mineralization assays, where each of the three antagonists tested provided a significant protective effect relative to dioxin alone (p < 0.05, Figure 7B,C). ANF demonstrated the strongest rescue effect on both cell migration and mineralization in dioxin-treated cells, with a full restoration to control levels; resveratrol and DIM also showed significant mitigating effects relative to dioxin-treated cells.

Discussion
Cigarette smoking greatly contributes to negative outcomes after spinal fusion procedures and rates of non-healing are significantly elevated in smokers (14% vs. 26%) [6]. Although smoking rates are on the decline, an estimated 20-25% of Americans still smoke and many more are subject to secondhand smoke [32]. These patients present a major dilemma for orthopedic surgeons who are severely limited in their ability to treat habitual smokers and who are left with the difficult choice of refusing surgical intervention or performing the procedure with significantly increased risks. A number of clinical studies have linked smoking with significant inhibitory effects on bone healing; however, inadequate clarification of the underlying molecular mechanisms has prevented the development of an appropriate therapeutic approach [33,34].
While it has been posited that a number of constituents of cigarette smoke-including nicotinemay play a role in the smoking-mediated inhibition of bone healing, the role of the aryl hydrocarbon receptor (AhR) has become a recent focus of active research [35,36]. The AhR is responsible for regulating xenobiotic metabolism and mediating the toxicity of environmental planar aromatic hydrocarbons, with previous studies suggesting deleterious effects of AhR hyper-activation on bone biology [12,13,37].
Dioxin, a minor hydrocarbon constituent of cigarette smoke but potent activator of the AhR, is thought to exert its effects almost exclusively through the receptor [20,21]. Because of its extremely high affinity for the receptor, dioxin is frequently used as a prototypical ligand to investigate the effects of the AhR pathway involvement in biological processes. Previous in vitro studies have shown that dioxin inhibits osteoblastic differentiation and ALP activity in murine cell lines and have also identified dioxin-sensitive proteins that are critical mediators of osteogenesis [12,38].

Discussion
Cigarette smoking greatly contributes to negative outcomes after spinal fusion procedures and rates of non-healing are significantly elevated in smokers (14% vs. 26%) [6]. Although smoking rates are on the decline, an estimated 20-25% of Americans still smoke and many more are subject to secondhand smoke [32]. These patients present a major dilemma for orthopedic surgeons who are severely limited in their ability to treat habitual smokers and who are left with the difficult choice of refusing surgical intervention or performing the procedure with significantly increased risks. A number of clinical studies have linked smoking with significant inhibitory effects on bone healing; however, inadequate clarification of the underlying molecular mechanisms has prevented the development of an appropriate therapeutic approach [33,34].
While it has been posited that a number of constituents of cigarette smoke-including nicotine-may play a role in the smoking-mediated inhibition of bone healing, the role of the aryl hydrocarbon receptor (AhR) has become a recent focus of active research [35,36]. The AhR is responsible for regulating xenobiotic metabolism and mediating the toxicity of environmental planar aromatic hydrocarbons, with previous studies suggesting deleterious effects of AhR hyper-activation on bone biology [12,13,37].
Dioxin, a minor hydrocarbon constituent of cigarette smoke but potent activator of the AhR, is thought to exert its effects almost exclusively through the receptor [20,21]. Because of its extremely high affinity for the receptor, dioxin is frequently used as a prototypical ligand to investigate the effects of the AhR pathway involvement in biological processes. Previous in vitro studies have shown that dioxin inhibits osteoblastic differentiation and ALP activity in murine cell lines and have also identified dioxin-sensitive proteins that are critical mediators of osteogenesis [12,38].
To confirm whether the AhR pathway is functional in MG-63 cells, we examined CYP1A1 expression, a well-established marker of AhR activation. Its induction by dioxin has confirmed that the AhR pathway is indeed intact in both non-differentiating and differentiating MG-63 cells. All three AhR antagonists reduced CYP1A1 expression to near baseline levels in dioxin-exposed cells (Figure 6), indicating that they are potent enough to achieve successful antagonism of the AhR in this setting.
Previous in vitro studies using both human and animal cell lines have explored changes in cell adhesion after dioxin treatment, as well as direct effects on integrins and cadherins in human pre-osteoblasts [38,39]. The reduced cellular adhesion rates we observed under both standard and osteogenic conditions may have implications for osteogenesis in vivo, where an inhibition of progenitor cell adhesion at the site of injury or scaffold implant could have adverse effects on the regenerative capacity (Figure 3). The observed reduction in cell adhesion rate and integrin expression levels demonstrates that dioxin may directly or indirectly modulates the expression or activity of adhesion-associated molecules, which could alter cell-cell and cell-matrix interactions. Further work should be done to determine if AhR antagonists can also mitigate the dioxin-mediated inhibition of cell adhesion and integrin expression.
CXCL12 is predominantly expressed by osteogenic stem and precursor cells prior to and immediately following the onset of osteogenesis, but its expression rapidly declines once cells commit to differentiation. Numerous groups have shown that CXCL12 signaling plays a role in the initiation of osteogenic differentiation. The interaction between CXCL12 and CXCR4 leads to both cytoskeletal rearrangement and migration of CXCR4-expressing cells toward a CXCL12 gradient [40]. Corresponding with our observed decrease in CXCL12 and CXCR4 mRNA expression, we found that dioxin exposure resulted in a decreased capacity for cell migration (Figures 4 and 6). Previous studies have shown CXCL12 upregulation at sites of injury, where it serves to recruit CXCR4-expressing stem cells in support of tissue-specific repair or regeneration [41]. Clinically, a reduction in progenitor cell motility could drastically impact the capacity for regeneration, since progenitors actively migrate towards an array of chemoattractants at the site of tissue injury. We postulate that dioxin-mediated downregulation of CXCL12 could play an important role in its adverse effects on bone regeneration in vivo, which will be explored in future work.
Notably, the expression levels of PHEX were significantly reduced in dioxin-treated differentiating MG-63 cells. PHEX is a zinc metalloendopeptidase that inhibits cleavage of the MEPE ASARM motif of SIBLING proteins (short integrin-binding ligand-interacting glycoproteins), which regulate mineralization. The released (free) ASARM peptide plays a major regulatory role in the mineralization of bones and teeth, as well as soft-tissue calcification, by binding strongly to hydroxyapatite and inhibiting mineralization [42]. We suspect that the downregulation of PHEX by dioxin-which would presumably result in an increase in levels of free ASARM-may lead to reduced mineral deposition, and this mechanism may be in part responsible for the decreased mineralization capacity and inhibition of bone healing seen in dioxin-exposed cells and animals.
With this work, we found that ALP activity is decreased after dioxin treatment but can be recovered with AhR antagonist co-treatment. The p38/ERK MAPK pathway, which is normally enhanced by BMP-2, plays an important role in osteoblastic differentiation and mineralization [43]. We found a decrease in overall p38/ERK and active p-p38/p-ERK levels ( Figure 4). This may in part explain the decreased ALP activity and mineralization in dioxin-treated cells. Whether p38 and ERK levels are recovered with AhR antagonist co-treatment will be examined in subsequent studies.
Overall, we observed that DIM (a selective AhR modulator), ANF (a partial AhR agonist/antagonist), and Res (a competitive AhR antagonist) worked similarly to promote osteogenesis in cells exposed to dioxin. Numerous AhR antagonists, such as resveratrol and DIM, can be found in an assortment of fruits and vegetables, as well as in over-the-counter dietary supplements. This class of compounds may represent a naturally sourced pharmacologic approach to combat the deleterious effects of cigarette smoke on bony healing. Bioavailability of these compounds is often low, making it difficult to achieve therapeutic concentrations with standard formulations currently on the market. Development of sustained-release formulations should be pursued to determine whether they can provide clinically significant protective effects.
Although our study delineates anti-osteogenic effects specific to dioxin, there are dozens of other ligand activators of the AhR present in cigarette smoke [9,43]. As such, these chemicals may have similar and potentially cumulative inhibitory effects on osteogenesis. The stability of these compounds varies drastically. For example, the half-life of dioxin is estimated to be 7 to 11 years, while the half-life of the polycyclic aromatic hydrocarbon, benzo[a]pyrene, is much shorter [14]. This may lead to variation in the impact that individual compounds have on the AhR and in turn bone healing. Identification of non-toxic compounds with antagonistic but not agonistic effects on the AhR is potentially of great medical interest and could improve the surgical outcomes of patients who smoke or have environmental or dietary exposure to AhR agonists.
Prior studies have focused on the isolated effects and mechanisms of specific chemical constituents present in cigarette smoke, but the cumulative effects of whole smoke on AhR activation and bone regeneration are not well understood. While it is important to appreciate the inhibitory role of individual chemicals such as dioxin on bone healing, the more clinically-relevant question is how the multitude of AhR ligands present in cigarette smoke collectively impact bone regeneration. Future studies should focus on clarifying the contribution of AhR activation by cumulative exposure to these various ligands contained within cigarette smoke to their inhibitory effects on bone regeneration and healing. There is also potential for additive or synergistic effects from treatment with multiple AhR antagonists, and this should be pursued in future work.
With a limited ability to treat patients who smoke and the low compliance with surgeons' requests for smoking cessation, an effective measure to reduce risk and improve patient outcomes would be extremely beneficial. This research identifies AhR antagonists that may provide a protective effect towards the harmful consequences of environmental contaminants present in cigarette smoke and other sources, and provides an argument for the development of therapeutics that target the AhR pathway to reduce the negative impact of cigarette smoke on bone.

MG-63 Cell Culture
Human MG-63 osteosarcoma cells, which are pre-osteoblast-like cells that differentiate into osteoblasts upon growth in osteoinductive media, were purchased from American Type Culture Collection (ATCC CRL-1427; Rockville, MD, USA). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution (standard media, SM), at 37 • C in a humidified atmosphere of 5% CO 2 . For all cell culture assays, media was replaced every 2-3 days. For induction of osteogenic differentiation, DMEM was supplemented with 15 mM β-glycerophosphate, 75 ng/mL L-ascorbic acid, and 15 nM dexamethasone (osteogenic media, OM). A dose of 100 nM dioxin was selected based on previous studies revealing 100% AhR binding saturation without cytotoxicity [44]. We also evaluated the effect of each antagonist on cell proliferation and found non-cytotoxic concentrations of α-naphthoflavone (ANF), resveratrol (Res), and 3,3 -Diindolylmethane (DIM) over the time period. These doses were therefore utilized for subsequent differentiation studies. Cells were treated with either vehicle control (dimethyl sulfoxide, DMSO; 0.1% final concentration) or the following: 100 nM dioxin, 0.5 µM ANF, 4 µM Res, and 10 µM DIM. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Alkaline Phosphatase Activity Assays
Cellular alkaline phosphatase (ALP) activity was quantified using a SensoLyte pNPP alkaline phosphate assay kit (Anaspec, Fermont, CA, USA). Cells were grown in standard or osteogenic media and treated with DMSO or dioxin for 7 days. Enzymatic reactions were performed on lysate supernatants as instructed by the manufacturer. A minimum of three independent experiments were performed for quantitation of ALP activity, as well as for all other in vitro assays.

Actin Filament Re-Polymerization Assay
MG-63 cells were directly seeded onto 8 µg/mL fibronectin-coated coverslips and treated with DMSO or dioxin for 3 days, followed by exposure to 1 µM of cytochalasin D for 1 h to depolymerize the actin cytoskeleton. After washing with PBS, cells were incubated with media to allow for actin re-polymerization. After 40 min, cells were fixed with 4% paraformaldehyde, blocked with 5% BSA for 1 h, and stained with rhodamine-conjugated phalloidin and DAPI for visualization of actin filament re-polymerization.

Cell Migration Assays
For wound healing assays, cells were maintained in "Culture-Insert 3 well plates" (ibidi, Madison, WI, USA) with DMSO-or dioxin-treated media. When cells reached confluence, the inserts were removed from the wells and the wells were washed twice with PBS. Cells were then incubated in DMSO-or dioxin-containing media for 15 h. Digital images were collected at time points 0, 8, 15, and 24 h using a light microscope in order to evaluate the rate of "wound closure." Transwell assays were performed to measure the effect of dioxin on directional cell migration capacity using previously published methods [19].

Mineralization Assays
Matrix mineralization was quantified using alizarin red staining followed by a cetylpyridinium chloride de-stain procedure, using previously published methodology [12]. After 3 min, cells were thoroughly washed and then de-stained with cetylpyridinium chloride. Absorbance of the de-stain solution was quantified using a Cytation 3 spectrophotometer at A 538nm (BioTek Instruments, Winooski, VT, USA). Mineralization was also confirmed by von Kossa staining by a previously established protocol [45].

RNA Isolation and Gene Expression Analysis
Primer sets for human genes used in this study are listed in Table 1. Primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Expression levels of mRNA were quantified using quantitative polymerase chain reaction (qPCR), which was performed using a Bio-Rad iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in the Equipment Core Facility of the Simpson Querrey Institute at Northwestern University. RNAsol, MinElute columns, complementary DNA (cDNA) synthesis kits, and 2X amfiSure qGreen qPCR Master Mix 2 were purchased by GenDepot (Barker, TX, USA). QPCR was performed using the following program: 94 • C denaturation for 5 min; then 40 repeated cycles of 94 • C, 45 s/55 • C, 1 min/68 • C for 1 min; 79 cycles at 55 • C for 30 s each for generation of melting curves. Relative gene expression was analyzed using the Livak method [46]. A normalized expression ratio (2 −∆∆Ct ) was calculated for each target gene, representing a fold difference in mRNA expression for treatment groups relative to vehicle control.

Statistical Analysis
Data were analyzed for overall statistical significance using a two-way ANOVA using Prism GraphPad (La Jolla, CA, USA). p-values less than 0.05 were regarded as statistically significant.