Microbially driven TLR5-dependent signaling governs distal malignant progression through tumor-promoting inflammation

The dominant TLR5(R392X) polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.Fil: Rutkowski, Melanie R.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Stephen, Tom L.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Svoronos, Nikolaos. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Allegrezza, Michael J.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Tesone, Amelia J.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Perales Puchalt, Alfredo. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Brencicova, Eva. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Escovar Fadul, Ximena. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Nguyen, Jenny M.. The Wistar Institute. Tumor Microenvironment and Metastasis Program; Estados UnidosFil: Cadungog, Mark G.. Christiana Care Health System. Helen F. Graham Cancer Center; Estados UnidosFil: Zhang, Rugang. The Wistar Institute. Gene Expression and Regulation Program; Estados UnidosFil: Salatino, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Tchou, Julia. University of Pennsylvania; Estados UnidosFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Conejo Garcia, Jose R.. The Wistar Institute. Gene Expression and Regulation Program; Estados Unido

Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of peritoneal fluid from patients with benign conditions.

<p>Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS or 100μg/ml polyI:C in the presence of 0%, 10% or 25% peritoneal fluid obtained from patients suffering from benign ovarian conditions. The following day, (A) the MFI of surface marker CD86 was assessed by flow cytometry and (B) cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 experiments (n = 12) were performed with DC from 8 different healthy volunteers cultured with ascites from 2 (n = 4) or 1 (n = 4) patient with benign ovarian conditions. One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test); * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.</p

Levels of various immunomodulatory factors in OC-associated ascites and their correlation with the immunosuppressive effects observed in DC activation assays.

<p>(A) Protein levels of various factors in ascites samples collected from OC patients were measured by sandwich ELISA. Eight malignant ascites samples used throughout the study are shown. Each symbol represents one patient sample throughout the graphs (n = 8). (B) Levels of IL-10 in ascites samples are correlated to the suppression of TLR-mediated up-regulation of CD86 and production of IL-6, IL-12 and TNFα. Suppression is expressed in per cent reduction of surface marker and cytokine levels when 10% ascites was added to the cell culture as compared to no ascites present; n = 26 (26 independent experiments conducted with monocyte-derived DC from 9 different healthy controls, cultured with ascites from two (n = 4), three (n = 2) or four (n = 3) different OC patients. The Pearson coefficient was calculated to assess statistical significance of correlations.</p

Neutralisation of IL-10 releases the impairment of DC activation in response to TLR stimulation in the presence of OC-associated ascites.

<p>(A, B) Monocyte derived DC were cultured in the presence of 3μg/ml R848, 10% ascites and neutralizing antibodies (5μg/ml) as indicated. Neutralizing antibodies were added to cultures alone or in combination. (A) CD86 expression levels were measured by flow-cytometry, and (B) cytokine levels were measured in cell culture supernatants by flow cytomix analysis (IL-6, TNFα) or sandwich ELISA (IL-12p40). n = 9–13 (13 independent experiments; monocyte-derived DC from 8 different healthy donors, cultured with 1 (n = 4), 2 (n = 3) or 3 (n = 1) different ascites samples. IL-6 neutralizing antibody was only used in 9 out of 13 experiments). One-way ANOVA (Friedman test with Dunn post test): *** = p<0.001. Please note that the quantification of IL-6 in samples containing IL-6 neutralizing antibody (αIL-6 and all AB) is compromised. (C, D) For allogeneic MLR, monocyte-derived DC from healthy donors were cultured overnight in complete medium only, or stimulated with 3μg/ml R848 in the presence or absence of 10% ascites fluid (AF) and/or IL-10 neutralizing antibody (5μg/ml) as indicated. Subsequently, such pre-treated DC were washed and co-cultured with CFSE-stained CD4⁺ naïve T cells from another healthy donor at a 1:200 ratio for 6 days. Naïve T cells from the same donor were used consistently for all experiments. The proliferation of T cells was determined by CFSE dilution after 6 days. (C) A representative example of flow-cytometry analysis is shown. (D) Cumulative data from 4 independent experiments showing proliferation in percent of CD3⁺ cells that have undergone at least one round of division. n = 4 (4 independent experiments; monocyte-derived DC from 3 different healthy donors, cultured with ascites from two (n = 1) or one (n = 2) OC patient).</p

Paracrine OC-associated PGE₂ impairs TLR-mediated DC activation and distinguishes malignant carcinoma from benign ovarian conditions.

<p>Monocyte-derived DC were stimulated overnight with 3μg/ml R848. (A-B) Malignant ascites and neutralizing antibodies (5μg/ml) against PGE₂ were added as indicated; n = 11 (11 independent experiments; monocyte-derived DC from 4 different healthy donors, cultured with 2 (n = 1), or 3 (n = 3) different ascites samples) (C) Neutralizing antibodies against IL-10 and PGE₂ were added as indicated to cultures containing 3μg/ml R848 and 10% ascites; n = 9 (9 independent experiments, monocyte-derived DC from 4 different healthy controls, cultured with 1 (n = 1), 2 (n = 1) or 3 (n = 2) different ascites samples). (D) Malignant ascites which had previously been depleted of PGE₂ (PGE2 ΔAF) or PGE₂ and IL-10 (PGE2/IL10 ΔAF) or which had undergone mock depletion (mock ΔAF) was added as indicated. As control, DC cultures were treated with R848 and untreated ascites (AF); n = 4 (4 independent experiments; monocyte-derived DC from 1 healthy donor, cultured with ascites samples from two individual ovarian carcinoma patients). The level of TNFα is expressed in relation to the TNFα levels induced in response to LPS, which were set to 100%. (A-D) Cytokine levels in cell culture supernatants were measured by sandwich ELISA. One-way ANOVA (Friedman test with Dunn post test); * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant. (E) PGE₂ levels in malignant ascites (n = 9) and benign peritoneal fluid (n = 6) were measured by sandwich ELISA. Mann-Whitney U test was used for statistical analysis.</p

Layering of stomach contents in drowning cases in post-mortem computed tomography compared to forensic autopsy

BACKGROUND In forensic autopsy, the analysis of stomach contents is important when investigating drowning cases. Three-layering of stomach contents may be interpreted as a diagnostic hint to drowning due to swallowing of larger amounts of water or other drowning media. The authors experienced frequent discrepancies of numbers of stomach content layering in drowning cases between post-mortem computed tomography (PMCT) and autopsy in forensic casework. Therefore, the goal of this study was to compare layering of stomach contents in drowning cases between PMCT and forensic autopsy. METHODS Drowning cases (n = 55; 40 male, 15 female, mean age 45.3 years; mean amount of stomach content 223 ml) that received PMCT prior to forensic autopsy were retrospectively analyzed by a forensic pathologist and a radiologist. Number of layers of stomach content in PMCT were compared to number of layers at forensic autopsy. RESULTS In 28 of the 55 evaluated drowning cases, a discrepancy between layering of stomach contents at autopsy compared to PMCT was observed: 1 layer at autopsy (n = 28): 50% discrepancy to PMCT, 2 layers (n = 20): 45% discrepancy, and 3 layers (n = 7): 71.4% discrepancy. Sensitivity of correctly determining layering (as observed at forensic autopsy) in PMCT was 52% (positive predictive value 44.8%). Specificity was 46.6% (negative predictive value 53.8%). In a control group (n = 35) of non-drowning cases, three-layering of stomach contents was not observed. CONCLUSION Discrepancies of observed numbers of stomach content layers between PMCT and forensic autopsy are a frequent finding possibly due to stomach content sampling technique at autopsy and movement of the corpse prior to PMCT and autopsy. Three-layering in PMCT, if indeed present, may be interpreted as a hint to drowning

Up-regulations of CD86 and induction of cytokines in response to TLR stimulation in the presence or absence of OC-associated ascites.

<p>(A) Monocyte-derived DC were stimulated overnight with 3μg/ml R848, 1μg/ml LPS, 100μg/ml polyI:C in the presence of 0%, 10% or 25% of ascites from patients with malignant OC. The mean fluorescence intensity (MFI) of the surface marker CD86 was assessed by flow cytometry. (B) Monocyte-derived DC were cultured overnight as described above and cytokines were measured in culture supernatants by flow cytomix analysis or sandwich ELISA (IL-12p40). In total, 12 independent experiments were performed (n = 12) with DC from individual healthy volunteers cultured with ascites from 4, 3, 2 or 1 OC patients (n = 1, 1, 1 and 3 healthy volunteers, respectively). One-way ANOVA was used for statistical analysis (Friedman test with Dunn post test): * = p<0.05; ** = p<0.01; *** = p<0.001; ns = not significant.</p