Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stressrelated clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

Inflammasomes are involved in gut homeostasis and inflammatory pathologies, but the role of NLRP3 inflammasome in these processes is not well understood. Cryopyrin-associated periodic syndrome (CAPS) patients with NLRP3 mutations have autoinflammation in skin, joints, and eyes, but not in the intestine. Here we show that the intestines of CAPS model mice carrying an Nlrp3 R258W mutation maintain homeostasis in the gut. Additionally, such mice are strongly resistant to experimental colitis and colorectal cancer; this is mainly through a remodelled gut microbiota with enhanced anti-inflammatory capacity due to increased induction of regulatory T cells (Tregs). Mechanistically, NLRP3R258W functions exclusively in the lamina propria mononuclear phagocytes to directly enhance IL-1β but not IL-18 secretion. Increased IL-1β boosts local antimicrobial peptides to facilitate microbiota remodelling. Our data show that NLRP3R258W-induced remodelling of the gut microbiota, induces local Tregs to maintain homeostasis and compensate for otherwise-detrimental intestinal inflammation.

The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor ®2 (TR®2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TR®2 direct a common precursor to a rod, M cone or S cone outcome using Nrlb2/b2 “knock-in” mice that express TR®2 instead of NRL from the endogenous Nrl gene. Nrlb2/b2 mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl−/− mice. Notably, the presence of both factors yielded rods in Nrl+/b2 mice. The results demonstrate innate plasticity in post-mitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRβ2. We also detected precursor cells in normal embryonic retina that transiently co-expressed Nrl and TRβ2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrlb2/b2 mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL and secondly, if NRL fails to act, M versus S cone identity dictated by TR®2.

Transforming growth factor-␤ (TGF-␤) is key in the pathogenesis of diabetic nephropathy. Thrombospondin 1 (TSP1) expression is increased in diabetes, and TSP1 regulates latent TGF-␤ activation in vitro and in diabetic animal models. Herein, we investigate the effect of blockade of TSP1-dependent TGF-␤ activation on progression of renal disease in a mouse model of type 1 diabetes (C57BL/6J-Ins2 Akita ) as a targeted treatment for diabetic nephropathy. Akita and control C57BL/6 mice who underwent uninephrectomy received 15 weeks of thrice-weekly i.p. treatment with 3 or 30 mg/kg LSKL peptide, control SLLK peptide, or saline. The effects of systemic LSKL peptide on dermal wound healing was assessed in type 2 diabetic mice (db/db). Proteinuria (urinary albumin level and albumin/creatinine ratio) was significantly improved in Akita mice treated with 30 mg/kg LSKL peptide. LSKL treatment reduced urinary TGF-␤ activity and renal phospho-Smad2/3 levels and improved markers of tubulointerstitial injury (fibronectin) and podocytes (nephrin). However, LSKL did not alter glomerulosclerosis or glomerular structure. LSKL did not increase tumor incidence or inflammation or impair diabetic wound healing. These data suggest that selective targeting of excessive TGF-␤ activity through blockade of TSP1-dependent TGF-␤ activation represents a therapeutic strategy for treating diabetic nephropathy that preserves the homeostatic functions of TGF-␤.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococccosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. In the current study, it was found that opsonization-mediated internalization of encapsulated C. neoformans also activated the canonical NLRP3–apoptosis-associated speck-like protein containing a CARD (ASC)–caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3–ASC–caspase-8 inflammasome as well, which resulted in robust IL-1β secretion and cell death from caspase-1–deficient primary dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans–induced activation of either the canonical NLRP3–ASC–caspase-1 inflammasome or the noncanonical NLRP3–ASC–caspase-8 inflammasome. Moreover, challenge with zymosan also led to the activation of the noncanonical NLRP3–ASC–caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection and suggest that manipulating NLRP3 signaling may help to control fungal challenge.

Based on the concept of the common mucosal immune system, immunization at various inductive sites can induce an immune response at other, remote mucosal surfaces. The immune responses elicited through rectal and oral routes of antigen delivery were compared with respect to (i) measurement of antibody responses in serum and various external secretions of the vaccinees and (ii) characterization of the nature and homing potentials of circulating antibody-secreting cells (ASC). Specific ASC appeared in the circulation in 4 of 5 volunteers after oral and 9 of 11 volunteers after rectal immunization withSalmonella typhi Ty21a. The kinetics, magnitude, and immunoglobulin isotype distribution of the ASC responses were similar in the two groups. In both groups, almost all ASC (99 or 95% after oral or rectal immunization, respectively) expressed α4β7, the gut homing receptor (HR), whereas l-selectin, the peripheral lymph node HR, was expressed only on 22 or 38% of ASC, respectively. Oral immunization elicited a more pronounced immune response in saliva and vaginal secretion, while rectal immunization was more potent in inducing a response in nasal secretion, rectum, and tears. No major differences were found in the abilities of the two immunization routes to induce a response in serum or intestinal secretion. Thus, the rectal antigen delivery should be considered as an alternative to the oral immunization route. The different immune response profiles found in various secretions after oral versus rectal antigen administration provide evidence for a compartmentalization within the common mucosal immune system in humans.

Transforming growth factor (TGF)-β supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-β to its biologically active form is a major regulatory node controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-β. TSP1 is increased in myeloma, and TSP1-TGF-β activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-β activity in myeloma and that antagonism of the TSP1-TGF-β axis inhibits myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-β that block TSP1-TGF-β activation were used to determine the role of the TSP1-TGF-β pathway in mouse models of myeloma. TSP1 binds to human myeloma cells and activates TGF-β produced by cultured human and mouse myeloma cell lines. Antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced TGF-β signaling (phospho-Smad 2) in bone sections, tumor burden, mouse IL-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. SRI31277 reduced tumor burden in the immune competent 5TGM1 myeloma model. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-β activation as a therapeutic strategy for targeted inhibition of TGF-β in myeloma.

Microsporum canis is a pathogenic fungus with worldwide distribution that causes tinea capitis in animals and humans. M. canis also causes invasive infection in immunocompromised patients. To defy pathogenic fungal infection, the host innate immune system is the first line of defense. As an important arm of innate immunity, the inflammasomes are intracellular multiprotein complexes that control the activation of caspase-1, which cleaves proinflammatory cytokine pro-interleukin-1␤ (IL-1␤) into its mature form. To determine whether the inflammasome is involved in the host defense against M. canis infection, we challenged human monocytic THP-1 cells and mouse dendritic cells with a clinical strain of M. canis isolated from patients with tinea capitis. We found that M. canis infection triggered rapid secretion of IL-1␤ from both THP-1 cells and mouse dendritic cells. Moreover, by using gene-specific shRNA and competitive inhibitors, we determined that M. canis-induced IL-1␤ secretion was dependent on NLRP3. The pathways proposed for NLRP3 inflammasome activation, namely, cathepsin B activity, K ؉ efflux, and reactive oxygen species production, were all required for the inflammasome activation triggered by M. canis. Meanwhile, Syk, Dectin-1, and Card9 were found to be involved in M. canis-induced IL-1␤ secretion via regulation of pro-IL-1␤ transcription. More importantly, our data revealed that M. canis-induced production of IL-1␤ was dependent on the NLRP3 inflammasome in vivo. Together, this study unveils that the NLRP3 inflammasome exerts a critical role in host innate immune responses against M. canis infection, and our data suggest that diseases that result from M. canis infection might be controlled by regulating the activation of inflammasomes.