SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts
Sun, J;Shin, DY;Eiseman, M;Yallowitz, AR;Li, N;Lalani, S;Li, Z;Cung, M;Bok, S;Debnath, S;Marquez, SJ;White, TE;Khan, AG;Lorenz, IC;Shim, JH;Lee, FS;Xu, R;Greenblatt, MB;
PMID: 34326333 | DOI: 10.1038/s41467-021-24819-w
Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.
Lui, JC;Raimann, A;Hojo, H;Dong, L;Roschger, P;Kikani, B;Wintergerst, U;Fratzl-Zelman, N;Jee, YH;Haeusler, G;Baron, J;
PMID: 35121733 | DOI: 10.1038/s41467-022-28318-4
SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. Homozygous loss-of-function mutations in SP7 cause osteogenesis imperfecta type XII, but neomorphic (gain-of-new-function) mutations of SP7 have not been reported in humans. Here we describe a de novo dominant neomorphic missense variant (c.926 C > G:p.S309W) in SP7 in a patient with craniosynostosis, cranial hyperostosis, and long bone fragility. Histomorphometry shows increased osteoblasts but decreased bone mineralization. Mice with the corresponding variant also show a complex skeletal phenotype distinct from that of Sp7-null mice. The mutation alters the binding specificity of SP7 from AT-rich motifs to a GC-consensus sequence (typical of other SP family members) and produces an aberrant gene expression profile, including increased expression of Col1a1 and endogenous Sp7, but decreased expression of genes involved in matrix mineralization. Our study identifies a pathogenic mechanism in which a mutation in a transcription factor shifts DNA binding specificity and provides important in vivo evidence that the affinity of SP7 for AT-rich motifs, unique among SP proteins, is critical for normal osteoblast differentiation.
Translatomic analysis of regenerating and degenerating spinal motor neurons in injury and ALS
Shadrach, J;Stansberry, W;Milen, A;Ives, R;Fogarty, E;Antonellis, A;Pierchala, B;
| DOI: 10.1016/j.isci.2021.102700
The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.
Different spatial distribution of inflammatory cells in the tumor microenvironment of ABC and GBC subgroups of diffuse large B cell lymphoma
Clinical and experimental medicine
Guidolin, D;Tamma, R;Annese, T;Tortorella, C;Ingravallo, G;Gaudio, F;Perrone, T;Musto, P;Specchia, G;Ribatti, D;
PMID: 33959827 | DOI: 10.1007/s10238-021-00716-w
Diffuse Large B-Cell Lymphoma (DLBCL) presents a high clinical and biological heterogeneity, and the tumor microenvironment chracteristics are important in its progression. The aim of this study was to evaluate tumor T, B cells, macrophages and mast cells distribution in GBC and ABC DLBCL subgroups through a set of morphometric parameters allowing to provide a quantitative evaluation of the morphological features of the spatial patterns generated by these inflammatory cells. Histological ABC and GCB samples were immunostained for CD4, CD8, CD68, CD 163, and tryptase in order to determine both percentage and position of positive cells in the tissue characterizing their spatial distribution. The results evidenced that cell patterns generated by CD4-, CD8-, CD68-, CD163- and tryptase-positive cell profiles exhibited a significantly higher uniformity index in ABC than in GCB subgroup. The positive-cell distributions appeared clustered in tissues from GCB, while in tissues from ABC such a feature was lower or absent. The combinations of spatial statistics-derived parameters can lead to better predictions of tumor cell infiltration than any classical morphometric method providing a more accurate description of the functional status of the tumor, useful for patient prognosis.
Rodríguez, JMM;Fonfara, S;Hetzel, U;Kipar, A;
PMID: 34955067 | DOI: 10.1177/03009858211062631
The sequence of pathological events in feline hypertrophic cardiomyopathy (fHCM) is still largely unknown, although we know that fHCM is characterized by interstitial remodeling in a macrophage-driven pro-inflammatory environment and that myocardial ischemia might contribute to its progression. This study aimed to gain further insights into the structural changes associated with interstitial remodeling in fHCM with special focus on the myocardial microvasculature and the phenotype of the interstitial cells. Twenty-eight hearts (16 hearts with fHCM and 12 without cardiac disease) were evaluated in the current study, with immunohistochemistry, RNA-in situ hybridization, and transmission electron microscopy. Morphometrical evaluations revealed a statistically significant lower microvascular density in fHCM. This was associated with structural alterations in capillaries that go along with a widening of the interstitium due to the accumulation of edema fluid, collagen fibers, and mononuclear cells that also proliferated locally. The interstitial cells were mainly of fibroblastic or vascular phenotype, with a substantial contribution of predominantly resident macrophages. A large proportion expressed CD34 mRNA, which suggests a progenitor cell potential. Our results indicate that microvascular alterations are key events in the pathogenesis of fHCM and that myocardial interstitial cell populations with CD34+ phenotype play a role in the pathogenesis of the disease.
International journal of molecular sciences
Son, M;Kim, GY;Yang, Y;Ha, S;Kim, J;Kim, D;Chung, HY;Moon, HR;Chung, KW;
PMID: 36902313 | DOI: 10.3390/ijms24054882
The peroxisome proliferator-activated receptor (PPAR) nuclear receptor has been an interesting target for the treatment of chronic diseases. Although the efficacy of PPAR pan agonists in several metabolic diseases has been well studied, the effect of PPAR pan agonists on kidney fibrosis development has not been demonstrated. To evaluate the effect of the PPAR pan agonist MHY2013, a folic acid (FA)-induced in vivo kidney fibrosis model was used. MHY2013 treatment significantly controlled decline in kidney function, tubule dilation, and FA-induced kidney damage. The extent of fibrosis determined using biochemical and histological methods showed that MHY2013 effectively blocked the development of fibrosis. Pro-inflammatory responses, including cytokine and chemokine expression, inflammatory cell infiltration, and NF-κB activation, were all reduced with MHY2013 treatment. To demonstrate the anti-fibrotic and anti-inflammatory mechanisms of MHY2013, in vitro studies were conducted using NRK49F kidney fibroblasts and NRK52E kidney epithelial cells. In the NRK49F kidney fibroblasts, MHY2013 treatment significantly reduced TGF-β-induced fibroblast activation. The gene and protein expressions of collagen I and α-smooth muscle actin were significantly reduced with MHY2013 treatment. Using PPAR transfection, we found that PPARγ played a major role in blocking fibroblast activation. In addition, MHY2013 significantly reduced LPS-induced NF-κB activation and chemokine expression mainly through PPARβ activation. Taken together, our results suggest that administration of the PPAR pan agonist effectively prevented renal fibrosis in both in vitro and in vivo models of kidney fibrosis, implicating the therapeutic potential of PPAR agonists against chronic kidney diseases.
Smart, CD;Fehrenbach, DJ;Wassenaar, JW;Agrawal, V;Fortune, NL;Dixon, DD;Cottam, MA;Hasty, AH;Hemnes, AR;Doran, AC;Gupta, DK;Madhur, MS;
PMID: 37314125 | DOI: 10.1093/cvr/cvad093
Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, microvascular dysfunction, and myocardial fibrosis with recent evidence implicating the immune system in orchestrating cardiac remodeling. Here, we show the mouse model of deoxycorticosterone acetate (DOCA)-salt hypertension induces key elements of HFpEF, including diastolic dysfunction, exercise intolerance, and pulmonary congestion in the setting of preserved ejection fraction. A modified single cell sequencing approach, CITE-seq, of cardiac immune cells reveals an altered abundance and transcriptional signature in multiple cell types, most notably cardiac macrophages. The DOCA-salt model results in differential expression of several known and novel genes in cardiac macrophages, including upregulation of Trem2, which has been recently implicated in obesity and atherosclerosis. The role of Trem2 in hypertensive heart failure, however, is unknown. We found that mice with genetic deletion of Trem2 exhibit increased cardiac hypertrophy, diastolic dysfunction, renal injury, and decreased cardiac capillary density after DOCA-salt treatment compared to wild-type controls. Moreover, Trem2-deficient macrophages have impaired expression of pro-angiogenic gene programs and increased expression of pro-inflammatory cytokines. Furthermore, we found that plasma levels of soluble TREM2 are elevated in DOCA-salt treated mice and humans with heart failure. Together, our data provide an atlas of immunological alterations that can lead to improved diagnostic and therapeutic strategies for HFpEF. We provide our dataset in an easy to explore and freely accessible web application making it a useful resource for the community. Finally, our results suggest a novel cardioprotective role for Trem2 in hypertensive heart failure.
Peisker, F;Halder, M;Nagai, J;Ziegler, S;Kaesler, N;Hoeft, K;Li, R;Bindels, EMJ;Kuppe, C;Moellmann, J;Lehrke, M;Stoppe, C;Schaub, MT;Schneider, RK;Costa, I;Kramann, R;
PMID: 35641541 | DOI: 10.1038/s41467-022-30682-0
The cardiac vascular and perivascular niche are of major importance in homeostasis and during disease, but we lack a complete understanding of its cellular heterogeneity and alteration in response to injury as a major driver of heart failure. Using combined genetic fate tracing with confocal imaging and single-cell RNA sequencing of this niche in homeostasis and during heart failure, we unravel cell type specific transcriptomic changes in fibroblast, endothelial, pericyte and vascular smooth muscle cell subtypes. We characterize a specific fibroblast subpopulation that exists during homeostasis, acquires Thbs4 expression and expands after injury driving cardiac fibrosis, and identify the transcription factor TEAD1 as a regulator of fibroblast activation. Endothelial cells display a proliferative response after injury, which is not sustained in later remodeling, together with transcriptional changes related to hypoxia, angiogenesis, and migration. Collectively, our data provides an extensive resource of transcriptomic changes in the vascular niche in hypertrophic cardiac remodeling.
Tuong, ZK;Loudon, KW;Berry, B;Richoz, N;Jones, J;Tan, X;Nguyen, Q;George, A;Hori, S;Field, S;Lynch, AG;Kania, K;Coupland, P;Babbage, A;Grenfell, R;Barrett, T;Warren, AY;Gnanapragasam, V;Massie, C;Clatworthy, MR;
PMID: 34936871 | DOI: 10.1016/j.celrep.2021.110132
The prostate gland produces prostatic fluid, high in zinc and citrate and essential for the maintenance of spermatozoa. Prostate cancer is a common condition with limited treatment efficacy in castration-resistant metastatic disease, including with immune checkpoint inhibitors. Using single-cell RNA-sequencing to perform an unbiased assessment of the cellular landscape of human prostate, we identify a subset of tumor-enriched androgen receptor-negative luminal epithelial cells with increased expression of cancer-associated genes. We also find a variety of innate and adaptive immune cells in normal prostate that were transcriptionally perturbed in prostate cancer. An exception is a prostate-specific, zinc transporter-expressing macrophage population (MAC-MT) that contributes to tissue zinc accumulation in homeostasis but shows enhanced inflammatory gene expression in tumors, including T cell-recruiting chemokines. Remarkably, enrichment of the MAC-MT signature in cancer biopsies is associated with improved disease-free survival, suggesting beneficial antitumor functions.
Herschke, F;Li, C;Zhu, R;Han, Q;Wu, Q;Lu, Q;Barale-Thomas, E;De Jonghe, S;Lin, TI;De Creus, A;
PMID: 34718044 | DOI: 10.1016/j.antiviral.2021.105196
JNJ-64794964 (JNJ-4964/AL-034/TQ-A3334), an oral toll-like receptor 7 agonist, is being investigated for the treatment of chronic hepatitis B (CHB), a condition with a high unmet medical need. The anti-hepatitis B (HBV) activity of JNJ-4964 was assessed preclinically in an adeno-associated virus vector expressing HBV (AAV/HBV) mouse model. Mice were treated orally with 2, 6 or 20 mg/kg of JNJ-4964 once-per-week for 12 weeks and then followed up for 4 weeks. At 6 mg/kg, a partial decrease in plasma HBV-DNA and plasma hepatitis B surface antigen (HBsAg) were observed, and anti-HBs antibodies and HBsAg-specific T cells were observed in 1/8 animals. At 20 mg/kg, plasma HBV-DNA and HBsAg levels were undetectable for all animals 3 weeks after start of treatment, with no rebound observed 4 weeks after JNJ-4964 treatment was stopped. High anti-HBs antibody levels were observed until 4 weeks after JNJ-4964 treatment was stopped. In parallel, HBsAg-specific immunoglobulin G-producing B cells and interferon-γ-producing CD4+ T cells were detected in the spleen. In 2/4 animals, liver HBV-DNA and HBV-RNA levels, and liver hepatitis B core antigen expression dropped 4 weeks after JNJ-4964 treatment-stop. In these animals, HBsAg-specific CD8+ T cells were detectable. Throughout the study, normal levels of alanine aminotransferase were observed, with no hepatocyte cell death (end of treatment and 4 weeks later) and minimal infiltrations of B and T cells into the liver, suggesting induction of cytokine-mediated, non-cytolytic mechanisms.
Maidji E, Moreno ME, Rivera JM, Joshi P, Galkina SA, Kosikova G, Somsouk M, Stoddart CA.
PMID: - | DOI: 10.3390/v11030256
Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.
Clinical science (London, England : 1979)
Kumar, R;Lee, MH;Kassa, B;Fonseca Balladares, DC;Mickael, C;Sanders, L;Andruska, A;Kumar, M;Spiekerkoetter, E;Bandeira, A;Stenmark, KR;Tuder, RM;Graham, BB;
PMID: 37014925 | DOI: 10.1042/CS20220642
Pulmonary hypertension (PH) can occur as a complication of schistosomiasis. In humans, schistosomiasis-PH persists despite antihelminthic therapy and parasite eradication. We hypothesized that persistent disease arises as a consequence of exposure repetition.Following intraperitoneal sensitization, mice were experimentally exposed to Schistosoma eggs by intravenous injection, either once or three times repeatedly. The phenotype was characterized by right heart catheterization and tissue analysis.Following intraperitoneal sensitization, a single intravenous Schistosoma egg exposure resulted in a PH phenotype that peaked at 7-14 days, followed by spontaneous resolution. Three sequential exposures resulted in a persistent PH phenotype. Inflammatory cytokines were not significantly different between mice exposed to one or three egg doses, but there was an increase in perivascular fibrosis in those who received three egg doses. Significant perivascular fibrosis was also observed in autopsy specimens from patients who died of this condition.Repeatedly exposing mice to schistosomiasis causes a persistent PH phenotype, accompanied by perivascular fibrosis. Perivascular fibrosis may contribute to the persistent schistosomiasis-PH observed in humans with this disease.
