Human α-Defensin 51-9 and Human ß-Defensin 2 Improve Metabolic Parameters and Gut Barrier Function in Mice Fed a Western-Style Diet.

Obesity and metabolic comorbidities are associated with gut permeability. While high-fructose and Western-style diet (WSD) disrupt intestinal barrier function, oral administration of human α-defensin 5 (HD5) and ß-defensin 2 (hBD2) is believed to improve intestinal integrity and metabolic disorders. Eighty-four male C57BL/6J mice were fed a WSD or a control diet (CD) ± fructose (F) for 18 weeks. In week 13, mice were randomly divided into three intervention groups, receiving defensin fragment HD51-9, full-length hBD2, or bovine serum albumin (BSA)-control for six weeks. Subsequently, parameters of hepatic steatosis, glucose metabolism, and gut barrier function were assessed. WSDF increased body weight and hepatic steatosis (p < 0.01) compared to CD-fed mice, whereas peptide intervention decreased liver fat (p < 0.05) and number of hepatic lipid droplets (p < 0.01) compared to BSA-control. In addition, both peptides attenuated glucose intolerance by reducing blood glucose curves in WSDF-fed mice. Evaluation of gut barrier function revealed that HD51-9 and hBD2 improve intestinal integrity by upregulating tight junction and mucin expression. Moreover, peptide treatment restored ileal host defense peptides (HDP) expression, likely by modulating the Wnt, Myd88, p38, and Jak/STAT pathways. These findings strongly suggest that α- and ß-defensin treatment improve hepatic steatosis, glucose metabolism, and gut barrier function.

Prefrontal allopregnanolone synergizes with D1 receptor activation to disrupt sensorimotor gating in male Sprague-Dawley rats.

RATIONALE: The prepulse inhibition (PPI) of the startle reflex is the best-established index of sensorimotor gating. We documented that the neurosteroid allopregnanolone (AP) is necessary to reduce PPI in response to D1 dopamine receptor agonists. Since Sprague-Dawley (SD) rats are poorly sensitive to the PPI-disrupting effects of these drugs, we hypothesized that AP might increase this susceptibility. OBJECTIVES: We tested whether AP is sufficient to increase the vulnerability of SD rats to PPI deficits in response to the D1 receptor full agonist SKF82958. METHODS: SD rats were tested for PPI after treatment with SKF82958 (0.05-0.3 mg/kg, SC) in combination with either intraperitoneal (1-10 mg/kg) or intracerebral (0.5 µg/µl/side) AP administration into the medial prefrontal cortex (mPFC) or nucleus accumbens shell. To rule out potential confounds, we measured whether SKF82958 affected the endogenous mPFC levels of AP. RESULTS: SD rats exhibited marked PPI deficits in response to the combination of systemic and intra-mPFC AP with SKF82958 but not with the D2 receptor agonist quinpirole (0.3-0.6 mg/kg, SC). SKF82958 did not elevate mPFC levels of AP but enhanced the content of its precursor progesterone. The PPI deficits caused by SKF82958 in combination with AP were opposed by the AP antagonist isoallopregnanolone (10 mg/kg, IP) and the glutamate NMDA receptor positive modulator CIQ (5 mg/kg, IP). CONCLUSION: These results suggest that AP enables the detrimental effects of D1 receptor activation on sensorimotor gating. AP antagonism or glutamatergic modulation counters these effects and may have therapeutic potential for neuropsychiatric disorders characterized by gating deficits.

Prefrontal allopregnanolone mediates the adverse effects of acute stress in a mouse model of tic pathophysiology.

Ample evidence suggests that acute stress can worsen symptom severity in Tourette syndrome (TS); however, the neurobiological underpinnings of this phenomenon remain poorly understood. We previously showed that acute stress exacerbates tic-like and other TS-associated responses via the neurosteroid allopregnanolone (AP) in an animal model of repetitive behavioral pathology. To verify the relevance of this mechanism to tic pathophysiology, here we tested the effects of AP in a mouse model recapitulating the partial depletion of dorsolateral cholinergic interneurons (CINs) seen in post-mortem studies of TS. Mice underwent targeted depletion of striatal CINs during adolescence and were tested in young adulthood. Compared with controls, partially CIN-depleted male mice exhibited several TS-relevant abnormalities, including deficient prepulse inhibition (PPI) and increased grooming stereotypies after a 30-min session of spatial confinement - a mild acute stressor that increases AP levels in the prefrontal cortex (PFC). These effects were not seen in females. Systemic and intra-PFC AP administration dose-dependently worsened grooming stereotypies and PPI deficits in partially CIN-depleted males. Conversely, both AP synthesis inhibition and pharmacological antagonism reduced the effects of stress. These results further suggest that AP in the PFC mediates the adverse effects of stress on the severity of tics and other TS-related manifestations. Future studies will be necessary to confirm these mechanisms in patients and define the circuitry responsible for the effects of AP on tics.

Acute stress impairs sensorimotor gating via the neurosteroid allopregnanolone in the prefrontal cortex.

Ample evidence indicates that environmental stress impairs information processing, yet the underlying mechanisms remain partially elusive. We showed that, in several rodent models of psychopathology, the neurosteroid allopregnanolone (AP) reduces the prepulse inhibition (PPI) of the startle, a well-validated index of sensorimotor gating. Since this GABAA receptor activator is synthesized in response to acute stress, we hypothesized its participation in stress-induced PPI deficits. Systemic AP administration reduced PPI in C57BL/6J mice and Long-Evans, but not Sprague-Dawley rats. These effects were reversed by isoallopregnanolone (isoAP), an endogenous AP antagonist, and the GABAA receptor antagonist bicuculline and mimicked by AP infusions in the medial prefrontal cortex (mPFC). Building on these findings, we tested AP's implication in the PPI deficits produced by several complementary regimens of acute and short-term stress (footshock, restraint, predator exposure, and sleep deprivation). PPI was reduced by acute footshock, sleep deprivation as well as the combination of restraint and predator exposure in a time- and intensity-dependent fashion. Acute stress increased AP concentrations in the mPFC, and its detrimental effects on PPI were countered by systemic and intra-mPFC administration of isoAP. These results collectively indicate that acute stress impairs PPI by increasing AP content in the mPFC. The confirmation of these mechanisms across distinct animal models and several acute stressors strongly supports the translational value of these findings and warrants future research on the role of AP in information processing.

Human ß-defensin 2 ameliorates acute GVHD by limiting ileal neutrophil infiltration and restraining T cell receptor signaling.

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human ß-defensin 2 (hBD-2) is an endogenous epithelial cell-derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal ß-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal ß-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Human ß-Defensin 2 Mutations Are Associated With Asthma and Atopy in Children and Its Application Prevents Atopic Asthma in a Mouse Model.

Asthma and allergies are complex, chronic inflammatory diseases in which genetic and environmental factors are crucial. Protection against asthma and allergy development in the context of farming environment is established by early animal contact, unpasteurized milk consumption and gut microbiota maturation. The human ß-defensin 2 (hBD-2) is a host defense peptide present almost exclusively in epithelial tissues, with pronounced immunomodulatory properties, which has recently been shown to ameliorate asthma and IBD in animal models. We hypothesized that adequate hBD-2 secretion plays a role in the protection against asthma and allergy development and that genetic variations in the complex gene locus coding for hBD-2 may be a risk factor for developing these diseases, if as a consequence, hBD-2 is insufficiently produced. We used MALDI-TOF MS genotyping, sequencing and a RFLP assay to study the genetic variation including mutations, polymorphisms and copy number variations in the locus harboring both genes coding for hBD-2 (DEFB4A and DEFB4B). We administered hBD-2 orally in a mouse model of house dust mite (HDM)-asthma before allergy challenge to explore its prophylactic potential, thereby mimicking a protective farm effect. Despite the high complexity of the region harboring DEFB4A and DEFB4B we identified numerous genetic variants to be associated with asthma and allergy in the GABRIELA Ulm population of 1,238 children living in rural areas, including rare mutations, polymorphisms and a lack of the DEFB4A. Furthermore, we found that prophylactic oral administration of hBD-2 significantly curbed lung resistance and pulmonary inflammation in our HDM mouse model. These data indicate that inadequate genetic capacity for hBD-2 is associated with increased asthma and allergy risk while adequate and early hBD-2 administration (in a mouse model) prevents atopic asthma. This suggests that hBD-2 could be involved in the protective farm effect and may be an excellent candidate to confer protection against asthma development.

Human ß-defensin-2 suppresses key features of asthma in murine models of allergic airways disease.

BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.

Human ß-Defensin 2 Mediated Immune Modulation as Treatment for Experimental Colitis.

Defensins represents an integral part of the innate immune system serving to ward off potential pathogens and to protect the intestinal barrier from microbial encroachment. In addition to their antimicrobial activities, defensins in general, and human ß-defensin 2 (hBD2) in particular, also exhibit immunomodulatory capabilities. In this report, we assessed the therapeutic efficacy of systemically administered recombinant hBD2 to ameliorate intestinal inflammation in three distinct animal models of inflammatory bowel disease; i.e., chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T-cell transfer into immunodeficient recipient mice. Treatment efficacy was confirmed in all tested models, where systemically administered hBD2 mitigated inflammation, improved disease activity index, and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Treatment of lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells with rhBD2 confirmed the immunomodulatory capacity in the circulatory compartment. Subsequent analyzes revealed dendritic cells (DCs) as the main target population. Suppression of LPS-induced inflammation was dependent on chemokine receptor 2 (CCR2) expression. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation. To our knowledge, this is the first study showing in vivo efficacy of a systemically administered defensin in experimental disease.

Isoallopregnanolone reduces tic-like behaviours in the D1CT-7 mouse model of Tourette syndrome.

Tourette syndrome (TS) is a neuropsychiatric disorder characterised by multiple, persistent tics. These semi-voluntary motor and phonic manifestations are typically aggravated by exposure to acute stress, yet the mechanisms underlying this exacerbation remain unclear. Using a well-characterised animal model of TS, the D1CT-7 mouse, we recently showed that acute stress increases tic-like responses and causes sensorimotor gating deficits, as measured by the prepulse inhibition of the startle. We showed that these effects are promoted by the brain synthesis of the neurosteroid allopregnanolone (AP). In line with this idea, inhibition of AP synthesis by finasteride was found to suppress the tic-exacerbating effects of stress; conversely, AP administration resulted in a marked enhancement of the number of tic-like motor bursts. Given that the primary mechanism of AP is based on the positive allosteric modulation of GABAA receptors, in the present study, we hypothesised that the enhancement in tic-like behaviours induced by either stress or AP may be countered by isoallopregnanolone (isoAP), the natural 3ß-epimer of AP that acts as an antagonist to the AP-binding site within GABAA receptors. In agreement with our hypothesis, isoAP (5-10 mg kg-1 , s.c.) dose-dependently reduced the number of tic-like behaviours induced by stress in D1CT-7 mice. These effects were comparable to those elicited by both the benchmark TS therapy haloperidol (0.3 mg kg-1 , i.p.), as well as finasteride (25 mg kg-1 , i.p.). IsoAP also countered the prepulse inhibition deficits secondary to stress in D1CT-7 mice. Finally, isoAP opposed the enhancement of tic-like behaviours induced by AP (15 mg kg-1 , i.p.). Given that isoAP is well-tolerated and has an optimal safety profile, these data suggest that this steroid may have therapeutic properties in TS.

Human Paneth cell α-defensin-5 treatment reverses dyslipidemia and improves glucoregulatory capacity in diet-induced obese mice.

Overnutrition is the principal cause of insulin resistance (IR) and dyslipidemia, which drive nonalcoholic fatty liver disease (NAFLD). Overnutrition is further linked to disrupted bowel function, microbiota alterations, and change of function in gut-lining cell populations, including Paneth cells of the small intestine. Paneth cells regulate microbial diversity through expression of antimicrobial peptides, particularly human α-defensin-5 (HD-5), and have shown repressed secretory capacity in human obesity. Mice were fed a 60% high-fat diet for 13 wk and subsequently treated with physiologically relevant amounts of HD-5 (0.001%) or vehicle for 10 wk. The glucoregulatory capacity was determined by glucose tolerance tests and measurements of corresponding insulin concentrations both before and during intervention. Gut microbiome composition was examined by 16S rRNA gene amplicon sequencing. HD-5-treated mice exhibited improved glucoregulatory capacity along with an ameliorated plasma and liver lipid profile. This was accompanied by specific decrease in jejunal inflammation and gut microbiota alterations including increased Bifidobacterium abundances, which correlated inversely with metabolic dysfunctions. This study provides proof of concept for the use of human defensins to improve host metabolism by mitigating the triad cluster of dyslipidemia, IR, and NAFLD.