Vascular endothelial profilin-1 drives a protumorigenic tumor microenvironment and tumor progression in renal cancer.

Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined in vivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) in vivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells in vivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.

STAT5 Represses a STAT3-Independent Th17-like Program during Th9 Cell Differentiation.

IL-9-producing Th cells, termed Th9 cells, contribute to immunity against parasites and cancers but have detrimental roles in allergic disease and colitis. Th9 cells differentiate in response to IL-4 and TGF-ß, but these signals are insufficient to drive Th9 differentiation in the absence of IL-2. IL-2-induced STAT5 activation is required for chromatin accessibility within Il9 enhancer and promoter regions and directly transactivates the Il9 locus. STAT5 also suppresses gene expression during Th9 cell development, but these roles are less well defined. In this study, we demonstrate that human allergy-associated Th9 cells exhibited a signature of STAT5-mediated gene repression that is associated with the silencing of a Th17-like transcriptional signature. In murine Th9 cell differentiation, blockade of IL-2/STAT5 signaling induced the expression of IL-17 and the Th17-associated transcription factor Rorγt. However, IL-2-deprived Th9 cells did not exhibit a significant Th17- or STAT3-associated transcriptional signature. Consistent with these observations, differentiation of IL-17-producing cells under these conditions was STAT3-independent but did require Rorγt and BATF. Furthermore, ectopic expression of Rorγt and BATF partially rescued IL-17 production in STAT3-deficient Th17 cells, highlighting the importance of these factors in this process. Although STAT3 was not required for the differentiation of IL-17-producing cells under IL-2-deprived Th9 conditions, their prolonged survival was STAT3-dependent, potentially explaining why STAT3-independent IL-17 production is not commonly observed in vivo. Together, our data suggest that IL-2/STAT5 signaling plays an important role in controlling the balance of a Th9 versus a Th17-like differentiation program in vitro and in allergic disease.

Reclassification of Treatment Strategy with Fractional Flow Reserve in Cancer Patients with Coronary Artery Disease.

Background and Objectives: Cancer and coronary artery disease (CAD) often coexist. Compared to quantitative coronary angiography (QCA), fractional flow reserve (FFR) has emerged as a more reliable method of identifying significant coronary stenoses. We aimed to assess the specific management, safety and outcomes of FFR-guided percutaneous coronary intervention (PCI) in cancer patients with stable CAD. Materials and Methods: FFR was used to assess cancer patients that underwent coronary angiography for stable CAD between September 2008 and May 2016, and were found to have ≥50% stenosis by QCA. Patients with lesions with an FFR > 0.75 received medical therapy alone, while those with FFR ≤ 0.75 were revascularized. Procedure-related complications, all-cause mortality, nonfatal myocardial infarction, or urgent revascularizations were analyzed. Results: Fifty-seven patients with stable CAD underwent FFR on 57 lesions. Out of 31 patients with ≥70% stenosis as measured by QCA, 14 (45.1%) had an FFR ≥ 0.75 and lesions were reclassified as moderate and did not receive PCI nor DAPT. Out of 26 patients with <70% stenosis as measured by QCA, 6 (23%) had an FFR < 0.75 and were reclassified as severe and were treated with PCI and associated DAPT. No periprocedural complications, urgent revascularization, acute coronary syndromes, or cardiovascular deaths were noted. There was a 22.8% mortality at 1 year, all cancer related. Patients who received a stent by FFR assessment showed a significant association with decreased risk of all-cause death (HR: 0.37, 95% CI 0.15−0.90, p = 0.03). Conclusions: Further studies are needed to define the optimal therapeutic approach for cancer patients with CAD. Using an FFR cut-off point of 0.75 to guide PCI translates into fewer interventions and can facilitate cancer care. There was an overall reduction in mortality in patients that received a stent, suggesting increased resilience to cancer therapy and progression.

Disruption of profilin1 function suppresses developmental and pathological retinal neovascularization.

Angiogenesis-mediated neovascularization in the eye is usually associated with visual complications. Pathological angiogenesis is particularly prominent in the retina in the settings of proliferative diabetic retinopathy, in which it can lead to permanent loss of vision. In this study, by bioinformatics analyses, we provide evidence for elevated expression of actin-binding protein PFN1 (profilin1) in the retinal vascular endothelial cells (VECs) of individuals with proliferative diabetic retinopathy, findings further supported by gene expression analyses for PFN1 in experimentally induced abnormal retinal neovascularization in an oxygen-induced retinopathy murine model. We observed that in a conditional knockout mouse model, postnatal deletion of the Pfn1 gene in VECs leads to defects in tip cell activity (marked by impaired filopodial protrusions) and reduced vascular sprouting, resulting in hypovascularization during developmental angiogenesis in the retina. Consistent with these findings, an investigative small molecule compound targeting the PFN1-actin interaction reduced random motility, proliferation, and cord morphogenesis of retinal VECs in vitro and experimentally induced abnormal retinal neovascularization in vivo In summary, these findings provide the first direct in vivo evidence that PFN1 is required for formation of actin-based protrusive structures and developmental angiogenesis in the retina. The proof of concept of susceptibility of abnormal angiogenesis to small molecule intervention of PFN1-actin interaction reported here lays a conceptual foundation for targeting PFN1 as a possible strategy in angiogenesis-dependent retinal diseases.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

Effects of Manganese Porphyrins on Cellular Sulfur Metabolism.

Manganese porphyrins (MnPs), MnTE-2-PyP5+, MnTnHex-2-PyP5+ and MnTnBuOE-2-PyP5+, are superoxide dismutase (SOD) mimetics and form a redox cycle between O2 and reductants, including ascorbic acid, ultimately producing hydrogen peroxide (H2O2). We previously found that MnPs oxidize hydrogen sulfide (H2S) to polysulfides (PS; H2Sn, n = 2-6) in buffer. Here, we examine the effects of MnPs for 24 h on H2S metabolism and PS production in HEK293, A549, HT29 and bone marrow derived stem cells (BMDSC) using H2S (AzMC, MeRho-AZ) and PS (SSP4) fluorophores. All MnPs decreased intracellular H2S production and increased intracellular PS. H2S metabolism and PS production were unaffected by cellular O2 (5% versus 21% O2), H2O2 or ascorbic acid. We observed with confocal microscopy that mitochondria are a major site of H2S production in HEK293 cells and that MnPs decrease mitochondrial H2S production and increase PS in what appeared to be nucleoli and cytosolic fibrillary elements. This supports a role for MnPs in the metabolism of H2S to PS, the latter serving as both short- and long-term antioxidants, and suggests that some of the biological effects of MnPs may be attributable to sulfur metabolism.

Controlled dimerization of insulin-like growth factor-1 and insulin receptors reveals shared and distinct activities of holo and hybrid receptors.

Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular ß subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.

Distribution of health facilities in Nigeria: Implications and options for Universal Health Coverage.

BACKGROUND: Nigeria is considering adopting Universal Health Coverage (UHC) as an official policy target to ensure access to quality health care services for her population without financial hardship. To facilitate discussion on the topic, the President of Nigeria convened a UHC summit in March 2014 to discuss Nigeria's options and strategies to achieve UHC. A strategy for achieving UHC requires analysis of the available infrastructure to deliver the services. We review the geographic and sectoral distribution of health facilities in Nigeria and discuss implications on the UHC strategy selected. METHODS: Secondary analysis of data from the Federal Ministry of Health's facility register was performed to assess the geographic and sectoral distribution of health facilities in Nigeria. Additionally, an extensive literature review was conducted to understand UHC strategies used by various countries and the associated health facility requirements. RESULTS: Primary health facilities make up 88% of health facilities in Nigeria while secondary and tertiary health facilities make up 12% and 0.25%, respectively. There are more government-owned health facilities than privately owned health facilities (67% vs 33%). Secondary health facilities are predominantly privately owned. The ratio of public to private health facilities is much higher in the northern part of the country than in the southern part. CONCLUSIONS: The distribution of health facilities across Nigeria is nonuniform. As such, a UHC strategy must be responsive to the variation in health facility distribution across the country. Additional investments are needed in some parts of the country to improve access to tertiary health facilities and leverage private sector capacity.

The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon.

OBJECTIVE: ATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohn's disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells. DESIGN: We genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant. RESULTS: We found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway. CONCLUSIONS: The CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.

Soluble bioactive microbial mediators regulate proteasomal degradation and autophagy to protect against inflammation-induced stress.

Bifidobacterium breve and other Gram-positive gut commensal microbes protect the gastrointestinal epithelium against inflammation-induced stress. However, the mechanisms whereby these bacteria accomplish this protection are poorly understood. In this study, we examined soluble factors derived from Bifidobacterium breve and their impact on the two major protein degradation systems within intestinal epithelial cells, proteasomes and autophagy. Conditioned media from gastrointestinal Gram-positive, but not Gram-negative, bacteria activated autophagy and increased expression of the autophagy proteins Atg5 and Atg7 along with the stress response protein heat shock protein 27. Specific examination of media conditioned by the Gram-positive bacterium Bifidobacterium breve (Bb-CM) showed that this microbe produces small molecules (<3 kDa) that increase expression of the autophagy proteins Atg5 and Atg7, activate autophagy, and inhibit proteasomal enzyme activity. Upregulation of autophagy by Bb-CM was mediated through MAP kinase signaling. In vitro studies using C2BBe1 cells silenced for Atg7 and in vivo studies using mice conditionally deficient in intestinal epithelial cell Atg7 showed that Bb-CM-induced cytoprotection is dependent on autophagy. Therefore, this work demonstrates that Gram-positive bacteria modify protein degradation programs within intestinal epithelial cells to promote their survival during stress. It also reveals the therapeutic potential of soluble molecules produced by these microbes for prevention and treatment of gastrointestinal disease.

Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity.

The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc-induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc-induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc-induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis.

Innate immune and chemically triggered oxidative stress modifies translational fidelity.

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Intestinal epithelial expression of TNFAIP3 results in microbial invasion of the inner mucus layer and induces colitis in IL-10-deficient mice.

Tumor necrosis factor-induced protein 3 (TNFAIP3; also known as A20) negatively regulates NF-κB and MAPK signals to control inflammatory responses. TNFAIP3 also protects against TNF-induced cell death. Intestinal epithelial cell (IEC) expression of TNFAIP3 improves barrier function and tight junction integrity and prevents dextran sulfate sodium (DSS)-induced IEC death and colitis. We therefore investigated the effects of TNFAIP3 expression in IEC on immune homeostasis in the intestines of immune-compromised mice. Villin-TNFAIP3 (v-TNFAIP3) transgenic mice were interbred with IL-10(-/-) mice (v-TNFAIP3 × IL-10(-/-)) and incidence, onset, and severity of colitis was assessed. v-TNFAIP3 × IL-10(-/-) mice displayed severe, early onset, and highly penetrant colitis that was not observed in IL-10(-/-) or v-TNFAIP3 mice. V-TNFAIP3 mice displayed altered expression of mucosal cytokines, increased numbers of mucosal regulatory T cells, and altered expression of mucosal antimicrobial peptides (AMPs). Microbial colonization of the inner mucus layer of v-TNFAIP3 mice was observed, along with alterations in the microbiome, but this was not sufficient to induce colitis in v-TNFAIP3 mice. The relative sterility of the inner mucus layer observed in wild-type and IL-10(-/-) mice was lost in v-TNFAIP3 × IL-10(-/-) mice. Thus IEC-derived factors, induced by signals that are inhibited by TNFAIP3, suppress the onset of inflammatory bowel disease in IL-10(-/-) mice. Our results indicate that IEC expression of TNFAIP3 alters AMP expression and allows microbial colonization of the inner mucus layer, which activates an IL-10-dependent anti-inflammatory process that is necessary to prevent colitis.

Egr1 mediates p53-independent c-Myc-induced apoptosis via a noncanonical ARF-dependent transcriptional mechanism.

c-Myc is frequently deregulated in human cancers. Although deregulated c-Myc leads to tumor growth, it also triggers apoptosis in partnership with tumor suppressors such as ARF and p53. Apoptosis induced by c-Myc is a critical fail-safe mechanism for the cell to protect against unrestrained proliferation. Despite the plethora of information on c-Myc, the molecular mechanism of how c-Myc induces both transformation and apoptosis is unclear. Oncogenic c-Myc can indirectly induce the expression of the tumor suppressor ARF, which leads to apoptosis through the stabilization of p53, but both c-Myc and ARF have apoptotic activities that are independent of p53. In cells without p53, ARF directly binds to c-Myc protein and inhibits c-Myc-induced hyperproliferation and transformation with a concomitant inhibition of canonical c-Myc target gene induction. However, ARF is an essential cofactor for p53-independent c-Myc-induced apoptosis. Here we show that ARF is necessary for c-Myc to drive transcription of a unique noncanonical target gene, Egr1. In contrast, c-Myc induces another family member, Egr2, through a canonical mechanism that is inhibited by ARF. We further demonstrate that Egr1 is essential for p53-independent c-Myc-induced apoptosis, but not ARF-independent c-Myc-induced apoptosis. Therefore, ARF binding switches the inherent activity of c-Myc from a proliferative to apoptotic protein without p53 through a unique noncanonical transcriptional mechanism. These findings also provide evidence that cofactors can differentially regulate specific transcriptional programs of c-Myc leading to different biological outcomes.

Intragenic Rearrangement Burden Associates with Immune Cell Infiltration and Response to Immune Checkpoint Blockade in Cancer.

Immune checkpoint blockade (ICB) can induce durable cancer remission. However, only a small subset of patients gains benefits. While tumor mutation burden (TMB) differentiates responders from nonresponders in some cases, it is a weak predictor in tumor types with low mutation rates. Thus, there is an unmet need to discover a new class of genetic aberrations that predict ICB responses in these tumor types. Here, we report analyses of pan-cancer whole genomes which revealed that intragenic rearrangement (IGR) burden is significantly associated with immune infiltration in breast, ovarian, esophageal, and endometrial cancers, particularly with increased M1 macrophage and CD8+ T-cell signatures. Multivariate regression against spatially counted tumor-infiltrating lymphocytes in breast, endometrial, and ovarian cancers suggested that IGR burden is a more influential covariate than other genetic aberrations in these cancers. In the MEDI4736 trial evaluating durvalumab in esophageal adenocarcinoma, IGR burden correlated with patient benefits. In the IMVigor210 trial evaluating atezolizumab in urothelial carcinoma, IGR burden increased with platinum exposure and predicted patient benefit among TMB-low, platinum-exposed tumors. Altogether, we have demonstrated that IGR burden correlates with T-cell inflammation and predicts ICB benefit in TMB-low, IGR-dominant tumors, and in platinum-exposed tumors.

Breast cancer cells promote osteoclast differentiation in an MRTF-dependent paracrine manner.

Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.

Retinoid orphan receptor gamma t (rorγt) promotes inflammatory eosinophilia but is dispensable for innate immune-mediated colitis.

Inflammatory bowel diseases (IBD) result from uncontrolled inflammation in the intestinal mucosa leading to damage and loss of function. Both innate and adaptive immunity contribute to the inflammation of IBD and innate and adaptive immune cells reciprocally activate each other in a forward feedback loop. In order to better understand innate immune contributions to IBD, we developed a model of spontaneous 100% penetrant, early onset colitis that occurs in the absence of adaptive immunity by crossing villin-TNFAIP3 mice to RAG1-/- mice (TRAG mice). This model is driven by microbes and features increased levels of innate lymphoid cells in the intestinal mucosa. To investigate the role of type 3 innate lymphoid cells (ILC3) in the innate colitis of TRAG mice, we crossed them to retinoid orphan receptor gamma t deficient (Rorγt-/-) mice. Rorγt-/- x TRAG mice exhibited markedly reduced eosinophilia in the colonic mucosa, but colitis persisted in these mice. Colitis in Rorγt-/- x TRAG mice was characterized by increased infiltration of the intestinal mucosa by neutrophils, inflammatory monocytes, macrophages and other innate cells. RNA and cellular profiles of Rorγt-/- x TRAG mice were consistent with a lack of ILC3 and ILC3 derived cytokines, reduced antimicrobial factors, increased activation oof epithelial repair processes and reduced activation of epithelial cell STAT3. The colitis in Rorγt-/- x TRAG mice was ameliorated by antibiotic treatment indicating that microbes contribute to the ILC3-independent colitis of these mice. Together, these gene expression and cell signaling signatures reflect the double-edged sword of ILC3 in the intestine, inducing both proinflammatory and antimicrobial protective responses. Thus, Rorγt promotes eosinophilia but Rorγt and Rorγt-dependent ILC3 are dispensable for the innate colitis in TRAG mice.

RNA-sequencing predicts a role of androgen receptor and aldehyde dehydrogenase 1A1 in osteosarcoma lung metastases.

One-third of pediatric patients with osteosarcoma (OS) develop lung metastases (LM), which is the primary predictor of mortality. While current treatments of patients with localized bone disease have been successful in producing 5-year survival rates of 65-70%, patients with LM experience poor survival rates of only 19-30%. Unacceptably, this situation that has remained unchanged for 30 years. Thus, there is an urgent need to elucidate the mechanisms of metastatic spread in OS and to identify targetable molecular pathways that enable more effective treatments for patients with LM. We aimed to identify OS-specific gene alterations using RNA-sequencing of extremity and LM human tissues. Samples of extremity and LM tumors, including 4 matched sets, were obtained from patients with OS. Our data demonstrate aberrant regulation of the androgen receptor (AR) pathway in LM and predicts aldehyde dehydrogenase 1A1 (ALDH1A1) as a downstream target. Identification of AR pathway upregulation in human LM tissue samples may provide a target for novel therapeutics for patients with LM resistant to conventional chemotherapy.

Vascular endothelial cell-specific disruption of the profilin1 gene leads to severe multiorgan pathology and inflammation causing mortality.

Actin-binding protein Profilin1 is an important regulator of actin cytoskeletal dynamics in cells and critical for embryonic development in higher eukaryotes. The objective of the present study was to examine the consequence of loss-of-function of Pfn1 in vascular endothelial cells (ECs) in vivo. We utilized a mouse model engineered for tamoxifen-inducible biallelic inactivation of the Pfn1 gene selectively in EC (Pfn1EC-KO). Widespread deletion of EC Pfn1 in adult mice leads to severe health complications presenting overt pathologies (endothelial cell death, infarct, and fibrosis) in major organ systems and evidence for inflammatory infiltrates, ultimately compromising the survival of animals within 3 weeks of gene ablation. Mice deficient in endothelial Pfn1 exhibit selective bias toward the proinflammatory myeloid-derived population of immune cells, a finding further supported by systemic elevation of proinflammatory cytokines. We further show that triggering Pfn1 depletion not only directly upregulates proinflammatory cytokine/chemokine gene expression in EC but also potentiates the paracrine effect of EC on proinflammatory gene expression in macrophages. Consistent with these findings, we provide further evidence for increased activation of Interferon Regulatory Factor 7 (IRF7) and STAT1 in EC when depleted of Pfn1. Collectively, these findings for the first time demonstrate a prominent immunological consequence of loss of endothelial Pfn1 and an indispensable role of endothelial Pfn1 in mammalian survival unlike tolerable phenotypes of Pfn1 loss in other differentiated cell types.