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Summary: Transforming growth factor β (TGF-β) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-β receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources.For complete details on the use and execution of this protocol, please refer to Garo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Activation of interleukin-17 (IL-17) receptor signaling within intestinal epithelial cells (IECs) promotes colorectal cancer development. Here, the authors show that miR-146a limits inflammation-induced colorectal carcinogenesis by inhibiting both IL-17 induction from myeloid cells and inhibiting IL-17R signaling within IECs.

A disequilibrium between immunosuppressive Tregs and inflammatory IL-17–producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying the Treg and Th17 imbalance in CNS autoimmunity remain largely unclear. Identifying the factors that drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. miR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell–intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg responses and dampening inflammatory T cell responses. Analogous to our findings in mice, miR-92a was elevated in CD4+ T cells from patients with MS, and miR-92a silencing in patients’ T cells promoted Treg development but limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.

Summary: Signal transduction and activator of transcription 3 (STAT3) is a key transcription factor implicated in the pathogenesis of kidney fibrosis. Although Stat3 deletion in tubular epithelial cells is known to protect mice from fibrosis, vFoxd1 cells remains unclear. Using Foxd1-mediated Stat3 knockout mice, CRISPR, and inhibitors of STAT3, we investigate its function. STAT3 is phosphorylated in tubular epithelial cells in acute kidney injury, whereas it is expanded to interstitial cells in fibrosis in mice and humans. Foxd1-mediated deletion of Stat3 protects mice from folic-acid- and aristolochic-acid-induced kidney fibrosis. Mechanistically, STAT3 upregulates the inflammation and differentiates pericytes into myofibroblasts. STAT3 activation increases migration and profibrotic signaling in genome-edited, pericyte-like cells. Conversely, blocking Stat3 inhibits detachment, migration, and profibrotic signaling. Furthermore, STAT3 binds to the Collagen1a1 promoter in mouse kidneys and cells. Together, our study identifies a previously unknown function of STAT3 that promotes kidney fibrosis and has therapeutic value in fibrosis.

Summary: Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-β on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-β inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer.

Summary: Splenic Ly6Chigh monocytes are innate immune cells involved in the regulation of central nervous system-related diseases. Recent studies have reported the shaping of peripheral immune responses by the gut microbiome via mostly unexplored pathways. In this study, we report that a 4-day antibiotic treatment eliminates certain families of the Bacteroidetes, Firmicutes, Tenericutes, and Actinobacteria phyla in the gut and reduces the levels of multiple pattern recognition receptor (PRR) ligands in the serum. Reduction of PRR ligands was associated with reduced numbers and perturbed function of splenic Ly6Chigh monocytes, which acquired an immature phenotype producing decreased levels of inflammatory cytokines and exhibiting increased phagocytic and anti-microbial abilities. Addition of PRR ligands in antibiotic-treated mice restored the number and functions of splenic Ly6Chigh monocytes. Our data identify circulating PRR ligands as critical regulators of the splenic Ly6Chigh monocyte behavior and suggest possible intervention pathways to manipulate this crucial immune cell subset.

After decades of development, inhibitors targeting cyclic nucleotide phosphodiesterases (PDEs) expressed in leukocytes have entered clinical practice for the treatment of inflammatory disorders, with three PDE4 inhibitors being in clinical use as therapeutics for psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease and atopic dermatitis. In contrast, the PDE8 family that is upregulated in pro-inflammatory T cells is a largely unexplored therapeutic target. We have previously demonstrated a role for the PDE8A-Raf-1 kinase complex in the regulation of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) activated CD4+ effector T cell adhesion and locomotion by a mechanism that differs from PDE4 activity. In this study, we explored the in vivo treatment of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS) induced in mice immunized with MOG using the PDE8-selective inhibitor PF-04957325. For treatment in vivo, mice with EAE were either subcutaneously (s.c.) injected three times daily (10 mg/kg/dose), or were implanted subcutaneously with Alzet mini-osmotic pumps to deliver the PDE8 inhibitor (15.5 mg/kg/day). The mice were scored daily for clinical signs of paresis and paralysis which were characteristic of EAE. We observed the suppression of the clinical signs of EAE and a reduction of inflammatory lesion formation in the CNS by histopathological analysis through the determination of the numbers of mononuclear cells isolated from the spinal cord of mice with EAE. The PDE8 inhibitor treatment reduces the accumulation of both encephalitogenic Th1 and Th17 T cells in the CNS. Our study demonstrates the efficacy of targeting PDE8 as a treatment of autoimmune inflammation in vivo by reducing the inflammatory lesion load.

Background: Ventricular fibrillation and atrial fibrillation are well-known arrhythmias in patients with Brugada syndrome. This study evaluated the characteristics of the atrial arrhythmogenic substrate using the signal-averaged electrogram (SAECG) in patients with Brugada syndrome. Methods: SAECGs were performed during normal sinus rhythm in 23 normal volunteers (control group), 21 patients with paroxysmal atrial fibrillation (PAF; PAF group), and 21 with Brugada syndrome (Brugada group). Results: The filtered P wave duration (fPd) in the control, Brugada, and PAF groups was 113.9 ± 12.9 ms, 125.3 ± 15.0 ms, and 137.1 ± 16.3 ms, respectively. The fPd in the PAF group was significantly longer compared to that in the control and Brugada groups (p

A 64 year-old man underwent percutaneous coronary intervention (PCI), and the conus branch was occluded. Ventricular fibrillation (VF) occurred five hours after the PCI procedure with a Brugada-like electrocardiogram. A continuous injection of amiodarone was effective for the suppression of VF.

Summary: Smad7, a negative regulator of TGF-β signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells. Smad7 deficiency in DCs promotes TGF-β responsiveness and the co-inhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs. In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro. The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-β and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention. : Smad7, a negative regulator of TGF-β signaling, is implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs). Here, Garo et al. describe how Smad7 within both dendritic cells and CD4+ T cells limits PD1-mediated Treg induction to promote intestinal inflammation, providing additional therapeutic strategies for IBD intervention. Keywords: Smad7, PD1, PDL1, PDL2, TGF-β, CD103, dendritic cell, DC, Treg, inflammatory bowel disease, IBD, ulcerative colitis, UC

Antipsychotics are often used in conjunction with anti-Alzheimer drugs to treat the behavioral and psychological symptoms of dementia (BPSD). Here, we examined the effects of cholinesterase inhibitors (ChEIs), donepezil and galantamine, on antipsychotic-induced extrapyramidal side effects (EPS) in mice. The effects of serotonergic agents on the EPS drug interaction were also evaluated. Donepezil (0.3–3 mg/kg) did not induce EPS signs by itself; however, it significantly potentiated bradykinesia induction with a low dose of haloperidol (0.5 mg/kg) in dose-dependent and synergistic manners. Galantamine (0.3–3 mg/kg) elicited mild bradykinesia at a high dose and dose-dependently augmented haloperidol-induced bradykinesia. The EPS potentiation by galantamine was blocked by trihexyphenidyl (a muscarinic antagonist), but not by mecamylamine (a nicotinic antagonist). In addition, the bradykinesia potentiation by galantamine was significantly reduced by (±)-8-hydroxy-2-(di-n-propylamino)-tetralin (a 5-HT1A agonist), ritanserin (a 5-HT2 antagonist), and SB-258585 (a 5-HT6 antagonist). The present results give us a caution for the antipsychotics and ChEIs interaction in inducing EPS in the treatment of BPSD. In addition, second generation antipsychotics, which can stimulate 5-HT1A receptors or antagonize 5-HT2 and 5-HT6 receptors, seem to be favorable as an adjunctive therapy for BPSD.

Unexplained cardiac arrest (UCA) with documented ventricular fibrillation (VF) is a major cause of sudden cardiac death. Abnormal sympathetic innervations have been shown to be a trigger of ventricular fibrillation. Further, adequate expression of SEMA3A was reported to be critical for normal patterning of cardiac sympathetic innervation. We investigated the relevance of the semaphorin 3A (SEMA3A) gene located at chromosome 5 in the etiology of UCA. Eighty-three Japanese patients diagnosed with UCA and 2,958 healthy controls from two different geographic regions in Japan were enrolled. A nonsynonymous polymorphism (I334V, rs138694505A>G) in exon 10 of the SEMA3A gene identified through resequencing was significantly associated with UCA (combined P = 0.0004, OR 3.08, 95%CI 1.67-5.7). Overall, 15.7% of UCA patients carried the risk genotype G, whereas only 5.6% did in controls. In patients with SEMA3A(I334V), VF predominantly occurred at rest during the night. They showed sinus bradycardia, and their RR intervals on the 12-lead electrocardiography tended to be longer than those in patients without SEMA3A(I334V) (1031±111 ms versus 932±182 ms, P = 0.039). Immunofluorescence staining of cardiac biopsy specimens revealed that sympathetic nerves, which are absent in the subendocardial layer in normal hearts, extended to the subendocardial layer only in patients with SEMA3A(I334V). Functional analyses revealed that the axon-repelling and axon-collapsing activities of mutant SEMA3A(I334V) genes were significantly weaker than those of wild-type SEMA3A genes. A high incidence of SEMA3A(I334V) in UCA patients and inappropriate innervation patterning in their hearts implicate involvement of the SEMA3A gene in the pathogenesis of UCA.