Ahmed Nadeem

Mucosal Allergic Sensitization to Cockroach Allergens Is Dependent on Proteinase Activity and Proteinase-Activated Receptor-2 Activation

We have shown that proteinase-activated receptor-2 (PAR2) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR2 in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR2. To study the role of PAR2 in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR2 activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR2, mice were exposed intranasally to a receptor-blocking anti-PAR2 Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR2 blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR2 activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR2-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR2 activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

Airway and systemic oxidant-antioxidant dysregulation in asthma: a possible scenario of oxidants spill over from lung into blood.

Asthma is characterized by repeated episodes of airway obstruction and an ongoing cycle of airway inflammation. Reactive oxygen species (ROS) produced by inflammatory cells in the lung play a key role in the pathogenesis as well as amplification of inflammation in asthmatic airways. Several enzymatic and non-enzymatic antioxidants are available in the lung and systemic circulation to counteract ROS-mediated damage on various biomolecules such as lipid membranes, proteins, and DNA; however during asthmatic inflammation these defenses are overwhelmed due to excessive production of ROS thereby leading to inflammatory events in the airways/systemic circulation. ROS-mediated damage may result in increased vascular permeability, mucus hypersecretion, smooth muscle contraction, epithelial shedding and impairment in the responsiveness of β-adrenergic receptors. Strategies aimed to boost the endogenous antioxidants either through dietary or pharmacological intervention to redress oxidant-antioxidant imbalance in asthma is the current area of research in many laboratories throughout the world. This review aims at providing a comprehensive overview of the available literature on oxidative stress and antioxidants imbalance in asthma with a focus both on lung and blood components and bring forth correlations between lung/blood oxidative stress/antioxidant parameters and lung function.

Regulation of TNF-α and NF-κB activation through the JAK/STAT signaling pathway downstream of histamine 4 receptor in a rat model of LPS-induced joint inflammation.

Histamine 4 receptor (H4R) is a novel target for the pharmacological modulation of histamine-mediated immune signals during inflammatory diseases. The purpose of this study was to assess the effects of the H4R agonist 4-methylhistamine dihydrochloride (4-MeH) and antagonist JNJ7777120 (JNJ) in the inflamed rat knee. Animals were fasted for 18h before a single dose of 4-MeH or JNJ (30mg/kg) was administered intraperitoneally (i.p.), both followed by intra-articular (i.a.) injection of LPS 2h later. Blood and synovial fluid were collected after a short incubation period and TNF-α, NF-κB, and IkB-α levels were measured via flow cytometry. Additionally, we assessed the effects of H4R engagement on the expression of IL-1β, TNF-α, and NF-κB mRNAs and the protein levels of TNF-α, NF-κB, JAK-1, and STAT-3 in the inflamed knee tissue. These results revealed increased TNF-α and NF-κB expression and decreased IkB-α levels in both the LPS alone and 4-MeH treated groups in whole blood and synovial fluid. Further, IL-1β, TNF-α, and NF-κB mRNA levels were significantly increased and western blot analysis confirmed increased expression of TNF-α, NF-κB, JAK-1, and STAT-3 in both LPS and 4-MeH treatment groups. Furthermore, these increases were completely inhibited in the inflamed knee tissue of the JNJ-treated group. Thus, the inhibition of inflammatory mediators and signaling pathways by the H4R antagonist JNJ suggests the anti-arthritic importance of this molecule.

Imiquimod-induced psoriasis-like skin inflammation is suppressed by BET bromodomain inhibitor in mice through RORC/IL-17A pathway modulation.

Psoriasis is one of the most common skin disorders characterized by erythematous plaques that result from hyperproliferative keratinocytes and infiltration of inflammatory leukocytes into dermis and epidermis. Recent studies suggest that IL-23/IL-17A/IL-22 cytokine axis plays an important role in the pathogenesis of psoriasis. The small molecule bromodomain and extraterminal domain (BET) inhibitors, that disrupt interaction of BET proteins with acetylated histones have recently demonstrated efficacy in various models of inflammation through suppression of several pathways, one of them being synthesis of IL-17A/IL-22 which primarily depends on transcription factor, retinoic acid receptor-related orphan receptor C (RORC). However, the efficacy and mechanistic aspect of a BET inhibitor in mouse model of skin inflammation has not been explored previously. Therefore, this study investigated the role of BET inhibitor, JQ-1 in mouse model of psoriasis-like inflammation. Mice were topically applied imiquimod (IMQ) to develop psoriasis-like inflammation on the shaved back and ear followed by assessment of skin inflammation (myeloperoxidase activity, ear thickness, and histopathology), RORC and its signature cytokines (IL-17A/IL-22). JQ-1 suppressed IMQ-induced skin inflammation as reflected by a decrease in ear thickness/myeloperoxidase activity, and RORC/IL-17A/IL-22 expression. Additionally, a RORα/γ agonist SR1078 was utilized to investigate the role of RORC in BET-mediated skin inflammation. SR1078 reversed the protective effect of JQ-1 on skin inflammation at both histological and molecular levels in the IMQ model. The current study suggests that BET bromodomains are involved in psoriasis-like inflammation through induction of RORC/IL-17A pathway. Therefore, inhibition of BET bromodomains may provide a new therapy against skin inflammation.

Dysregulation of Th1, Th2, Th17, and T regulatory cell-related transcription factor signaling in children with autism

Autism is a neurodevelopmental disorder characterized by stereotypic repetitive behaviors, impaired social interactions, and communication deficits. Numerous immune system abnormalities have been described in individuals with autism including abnormalities in the ratio of Th1/Th2/Th17 cells; however, the expression of the transcription factors responsible for the regulation and differentiation of Th1/Th2/Th17/Treg cells has not previously been evaluated. Peripheral blood mononuclear cells (PBMCs) from children with autism (AU) or typically developing (TD) control children were stimulated with phorbol-12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A. The expressions of Foxp3, RORγt, STAT-3, T-bet, and GATA-3 mRNAs and proteins were then assessed. Our study shows that children with AU displayed altered immune profiles and function, characterized by a systemic deficit of Foxp3+ T regulatory (Treg) cells and increased RORγt+, T-bet+, GATA-3+, and production by CD4+ T cells as compared to TD. This was confirmed by real-time PCR (RT-PCR) and western blot analyses. Our results suggest that autism impacts transcription factor signaling, which results in an immunological imbalance. Therefore, the restoration of transcription factor signaling may have a great therapeutic potential in the treatment of autistic disorders.

Proteinase activated receptor-2-mediated dual oxidase-2 up-regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma.

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase‐activated receptor 2 (PAR‐2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX‐2, have been shown to be involved in airway inflammation in response to Toll‐like receptor activation. However, the association between PAR‐2 and DUOX‐2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX‐2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR‐2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX‐2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl‐2, caspase‐3). Challenge with CE led to up‐regulation of DUOX‐2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD‐1068, a small molecule antagonist of PAR‐2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX‐2/ROS pathway; however, these effects were less pronounced than PAR‐2 antagonism. The current study suggests that PAR‐2 activation leads to up‐regulation of the DUOX‐2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.

Carbon tetrachloride-induced hepatotoxicity in rat is reversed by treatment with riboflavin

Liver is a vital organ for the detoxification of toxic substances present in the body and hepatic injury is associated with excessive exposure to toxicants. The present study was designed to evaluate the possible hepatoprotective effects of riboflavin against carbon tetrachloride (CCl4) induced hepatic injury in rats. Rats were divided into six groups. Hepatotoxicity was induced by the administration of a single intraperitoneal dose of CCl4 in experimental rats. Riboflavin was administered at 30 and 100mg/kg by oral gavage to test its protective effect on hepatic injury biochemically and histopathologically in the blood/liver and liver respectively. The administration of CCl4 resulted in marked alteration in serum hepatic enzymes (like AST, ALT and ALP), oxidant parameters (like GSH and MDA) and pro-inflammatory cytokine TNF-α release from blood leukocytes indicative of hepatic injury. Changes in serum hepatic enzymes, oxidant parameters and TNF-α production induced by CCl4 were reversed by riboflavin treatment in a dose dependent manner. Treatment with standard drug, silymarin also reversed CCl4 induced changes in biomarkers of liver function, oxidant parameters and inflammation. The biochemical observations were paralleled by histopathological findings in rat liver both in the case of CCl4 and treatment groups. In conclusion, riboflavin produced a protective effect against CCl4-induced liver damage. Our study suggests that riboflavin may be used as a hepato-protective agent against toxic effects caused by CCl4 and other chemical agents in the liver.

Histamine 4 receptor promotes expression of costimulatory B7.1/B7.2 molecules, CD28 signaling and cytokine production in stress-induced immune responses.

Recently, the expression of histamine 4 receptor (H4R) on neurons was reported, however its function in cells within the central nervous system (CNS) remains poorly understood. To this end, we used the H4R agonist, 4-methylhistamine (4-MeH), and the H4R antagonist, JNJ77777120 (JNJ), to investigate the function of H4R signaling in immune cells in a murine model of chronic stress. Treatment of stressed mice with 4-MeH resulted in an increase in the proportion of lymphocyte subsets (CD3(+), CD8(+), CD28(+), and CD4(+)CD28(+)) and cells expressing the co-stimulatory molecules CD80(+) (B7.1) and CD86(+) (B7.2) in heparinized blood as compared to normal control (NC) and stressed control (SC) groups. We also observed that as compared to NC and SC mice, 4-MeH-treated mice showed greater production of IL-2(+), IL-6(+), IL-9(+), IL-21(+), and IL-27(+) cytokines in the spleen and by splenic CD4(+) T cells. Furthermore, 4-MeH treatment of stressed mice led to an increase in the levels of serum Th1/Th17 cytokines and corticosterone, and a decrease in Th2 cytokines. Treatment of chronically-stressed mice with 4-MeH also augmented expression of IL-6, IL-21, NF-κB p65, and STAT3 mRNA. Moreover, Western blot analyses confirmed increased protein expression of NF-κB, iNOS, and STAT3 expression following 4-MeH treatment of chronically-stressed mice as compared to controls. These proteins provide a novel relevant targets for the manipulation of chronic stress induced immune regulation. In striking contrast, treatment of stressed mice with the H4R antagonist, JNJ, resulted in a substantial reduction in all of the aforementioned effects upon immune cell percentages and cytokine production.

STA-21, a STAT-3 inhibitor, attenuates the development and progression of inflammation in collagen antibody-induced arthritis

We set out to investigate the influence of STA-21, a dynamic STAT-3 inhibitor, on the expansion and progression of rheumatoid arthritis (RA), and to determine its potential mechanisms of action in a mouse model of collagen antibody-induced arthritis (CAIA). To this end, arthritis was induced via intravenous (IV) injection of Balb/c mice with a cocktail of antibodies directed against type II collagen (1.5μg/mouse, IV), followed by lipopolysaccharide (LPS) at a dose of (25μg/mouse, i.p.) on day 3. Mice were then left untreated or were simultaneously treated with STA-21 (0.5mg/kg, i.p., once daily for 2 weeks) followed by evaluation for clinical and histological features of arthritic inflammation and flow cytometric analysis of cytokines and transcription factors in peripheral blood. STA-21 enhanced the clinical course of arthritis in CAIA mice and decreased CD8+RORγt+ and CD8+IL-21+ cells while inducing the production of CD8+Foxp3+ cells. Furthermore, STA-21 prevented the production of TNF-α and IL-6 in peripheral blood and increased IL-27 production by CD14+ cells. Moreover, STA-21 not only regulates Th1/Th2 serum cytokine levels but also the mRNA and protein expression of key factors including NF-κB p65, RORγt, T-bet, IL-4, GATA-3, JAK1, Stat3, and IL-21. Thus, administration of the Stat3 inhibitor STA-21 inhibits cellular signaling pathways and downstream activation of key transcription factors previously shown to play key roles in the pathogenesis of RA. Therefore, these data suggest that STA-21 could be considered as a potential treatment for patients with RA.

Resveratrol treatment attenuates chemokine receptor expression in the BTBR T + tf/J mouse model of autism

Autism is a neurodevelopmental disorder categorized by qualitative impairments in social interaction, communication, and repetitive stereotypic behavior. Emerging evidence increasingly suggests that chemokine receptors have a pivotal role in the central nervous system and are involved in the pathogenesis of numerous neuroinflammatory diseases. Resveratrol is widely used to treat neurodegenerative diseases, but its effect on autism has not been investigated. We investigated the effect of resveratrol (20 and 40mg/kg) in the spleen and brain tissues of BTBR T+tf/J (BTBR) and C57BL/6J (B6) mice as well as on the C-C chemokine receptor (CCR) and C-X-C motif chemokine receptor (CXCR) (CCR3+, CCR5+, CCR7+ and CCR9+, CXCR3+ and CXCR5+) in cluster of differentiation 4-positive (CD4+) T cells in the spleen. We also assessed the mRNA expression of CCR and CXCR receptors in the spleen and brain tissues. Our study revealed that the BTBR and B6 control mice showed different immune profiles. The BTBR mice showed characteristic higher levels of both CCR and CXCR production and expression in CD4+ T cells than the B6 control mice did. Treatment of B6 and BTBR mice with resveratrol (20 and 40mg/kg) induced a substantial decrease in the CCR and CXCR production and expression in CD4+ T cells compared with the respective untreated control groups. Moreover, resveratrol treatment decreased the mRNA expression levels of CCR and CXCR in the spleen and brain tissues. Resveratrol downregulated the chemokine receptor levels, which might provide unique targets for future therapies for autism.