COMMON AND DIFFERENTIAL ROLES OF INDUCIBLE NO SYNTHASE AND POLY (ADP-RIBOSE) POLYMERASE IN ALLERGEN-INDUCED INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS: A POTENTIAL CONNECTION TO NO LEVELS

Asthma is a chronic disease of the airways characterized by inflammation, structural and functional changes that are responsible for bronchial hyperresponsiveness (AHR) and limitation in airflow. While asthma symptoms can be controlled in the majority of patients, in around 5-10% of asthmatics, the disease remains symptomatic and inadequately controlled. For these reasons, additional therapeutic approaches are urgently required to provide better life conditions for these individuals. The present study is focused on the role of inducible Nitric Oxide Synthase (iNOS) and poly(ADP-ribose) polymerase-1 (PARP-1) in the pathophysiology of asthma and their potential interaction and cooperation. The role of iNOS in asthma has been examined in numerous studies with conflicting results. Its potential as a viable therapeutic target for the treatment of the disease was severely hampered by the negative results of a clinical trial showing that a selective iNOS inhibitor, although reducing effectively exhaled Nitric Oxide (eNO), did not affect AHR or airway inflammation after allergen challenge in human subjects with asthma. We believe that such conclusion is premature and that more careful studies are necessary to fully understand the role of iNOS in asthma and whether the enzyme can be adequately targeted at least as an adjuvant therapy for the treatment of this disease. In the current study I report that iNOS inhibition, pharmacologically, using the relatively selective inhibitor L-NIL or by gene knockout, in our animal experimental models provides an excellent protection against AHR upon an acute, but not upon a chronic, exposure to allergens (OVA or HDM) as assessed using full body plethysmography. These results correlate with the differential protection provided by iNOS inhibition against airway inflammation observed in a previous study. Considering all known beneficial effects of NO through its contribution to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium, I speculate that complete inhibition of iNOS, achieved by L-NIL administration or even more efficiently by gene knockout may be deleterious for some aspects of asthma such as AHR, which highly involve one of the major targets of NO, the smooth muscle cells. The fascinating aspect of my study is that the loss of protection observed in the chronic asthma model after L-NIL treatment was reversed by NO supplementation through administration of nitrite, a moderate NO source. The anion nitrite (NO2\u2013) can be reduced to NO upon a reaction with deoxyhemoglobin. Our group, in previous studies, also established a reciprocal relationship between iNOS and PARP-1 with a special connection to oxidative DNA damage and IL-5 in the process of eosinophilia during allergen-induced lung inflammation. Such relationship is also based on the fact that PARP-1 exerts a decisive role on the interaction between p65 NF-\u3baB and the exportin protein Crm1. PARP-1 activity, decrease the interaction between the transcription factor and Crm1, increasing the quantity of p65 NF-\u3baB detectable in the nuclei of cells upon TLR4 stimulation. This increase leads to an upregulation of NF-\u3baB\u2013dependent gene expression, which includes iNOS. PARP inhibition, pharmacologically by Olaparib (AZD2281) or by gene knockout, protects against inflammation and AHR both upon single and multiple exposures to OVA. Similar protection was observed upon chronic exposure to HDM, demonstrating that the protective effect is not limited to the type of allergen used. Such protection may occur as a consequence that iNOS expression is markedly reduced upon PARP inhibition. However, it is interesting that PARP-1 inhibition does not completely abrogate expression of iNOS leaving the possibility that the protective effect of PARP inhibition against inflammation and AHR may be associated with the reduction but not the complete inhibition of iNOS and with consequent production of moderate levels of NO. A further confirmation of our hypothesis is that administration of iNOS inhibitor (L-NIL) to chronically HDM-exposed mice that received a PARP inhibitor (Olaparib-AZD2281) reversed the protection against AHR provided by PARP inhibition. Overall, our results may explain why clinical studies focused on the complete inhibition of iNOS failed to protect against different aspects of asthma. However, further efforts to investigate the exact role of the enzyme in asthma may provide a clearer view on how to utilize iNOS as a therapeutic target. But what seems to be plausible is that moderate levels of NO achieved through partial inhibition of iNOS could represent a good therapeutic strategy at least against some aspect of the disease, such as AHR. Additionally, our results revealed that targeting PARP-1 may constitute a better alternative therapeutic approach. Such strategy seems to be very efficient at reducing the most important asthma traits (e.g. Th2 cytokines, mucus, and IgE production) but also partially reduces expression of iNOS with a consequent moderate production of NO that provides protections against AHR. It is important to note that the beneficial effects of PARP inhibition could be correlated with the capacity of PARP-1 to positively influence GATA3, the mast regulator of Th2 population, the paramount cells in the manifestation of the allergic disease. This is, of course, without omitting the potential impact of PARP inhibition on the T-reg population, which is responsible for the control and reduction of inflammation. Finally, it is noteworthy that L-NIL (L-N6-(1-Iminoethyl)lysine dihydrochloride) and AZD2281 (olaparib) are two clinically tested iNOS and PARP inhibitors, respectively, which increases considerably the clinical relevance of this study

HPV in sperm is efficiently removed by washing: A suitable approach for assisted reproduction

Research question: Is it possible, by sperm-washing spermatozoa from clinically HPV-positive men, to obtain spermatozoa free of human papillomavirus (HPV) to be employed in assisted reproduction? Design: This was an observational study performed on HPV-positive men. Freshly ejaculated semen was collected and readily processed by gradient separation followed by swim-up from the washed pellet. The resulting fractions were seminal plasma, cell pellet, round cells, non-motile spermatozoa and motile spermatozoa. All fractions were then tested for the presence of HPV DNA. Results: Of the 15 clinically HPV-positive subjects, 67% were positive in at least one of the seminal fractions. If any postivity was detected, the plasma was always HPV positive. No consistent pattern was observed throughout different samples in the cell pellet, round cell and non-motile spermatozoa fractions. However, after the sperm-wash procedure, the fraction of motile spermatozoa was never found to be HPV-positive. Conclusions: The sperm-washing technique, which was previously successfully used to remove human immunodeficiency virus, can efficiently remove HPV from spermatozoa. However, the present study was conducted on a small population so a larger follow-up study is recommended. HPV screening should be performed in sperm samples and, upon HPV positivity, sperm-washing should be considered before assisted reproduction techniques are used

Sterol metabolism modulates susceptibility to HIV-1 Infection

Background: 25-hydroxylase (CH25H) is an Interferon stimulated gene (ISG), which catalyzes the synthesis of 25-Hydroxycholesterol (25HC). 25HC intervenes in metabolic and infectious processes as controls cholesterol homeostasis and influences viral entry into host cells.We verified whether natural resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) individuals is at least partially mediated by particularities in sterol biosynthesis. Methods: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) isolated from 15 sexually-exposed HESN and 15 healthy controls (HC) were in vitro HIV-1-infected and analyzed for: 1) percentage of IFN\u3b1-producing plasmacytoid Dendritic Cells (pDCs); 2) Cholesterol signaling and inflammatory response RNA expression; 3) resistance to HIV-1 infection. MDMs from 5 HC were in vitro HIV-1-infected in the absence/presence of exogenously added 25HC. Results: IFN\u3b1-producing pDCs were augmented in HESN compared to HCs both in unstimulated and in in vitro HIV-1-infected PBMCs (p<0.001). An increased expression of CH25H and of a number of genes involved in cholesterol metabolism (ABCA1, ABCG1, CYP7B1, LXR\u3b1, OSBP, PPAR\u3b3, SCARB1) was observed as well; this, was associated with a reduced susceptibility to in vitro HIV-1-infection of PBMCs and MDMs (p<0.01). Notably, addition of 25HC to MDMs resulted in increased cholesterol efflux and augmented resistance to in vitro HIV-1-infection. Conclusions: Results herein show that in HESN sterol metabolism might be particularly efficient. This could be related to the activation of the IFN\u3b1 pathway and results into a reduced susceptibility to in vitro HIV-1 infection. These results suggest a possible basis for therapeutic interventions to modulate HIV-1 infection

Endoplasmic Reticulum Associated Aminopeptidase 2 (ERAP2) is released in the secretome of activated MDMs and reduces in vitro HIV-1 infection

Background: Haplotype-specific alternative splicing of the endoplasmic reticulum (ER) aminopeptidase type 2 (ERAP2) gene results in either full-length (FL, haplotype A) or alternatively spliced (AS, haplotype B) mRNA. HapA/HapA homozygous (HomoA) subjects show a reduced susceptibility to HIV-1 infection, probably secondary to the modulation of the antigen processing/presenting machinery. ERAP1 was recently shown to be secreted from the plasma membrane in response to activation; we investigated whether ERAP2 can be released as well and if the secreted form of this enzyme retains its antiviral function. Methods: Human monocyte derived macrophages (MDMs) were differentiated from peripheral blood mononuclear cells (PBMCs) isolated from 6 HomoA healthy controls and stimulated with IFN\u3b3 and LPS. ERAP2-FL secretion was evaluated by mass spectrometry. PBMCs (14 HomoA and 16 HomoB) and CD8-depleted PBMCs (CD8-PBMCs) (4 HomoA and 4 HomoB) were in vitro HIV-infected in the absence/presence of recombinant human ERAP2-FL (rhERAP2) protein; p24 viral antigen quantification was used to assess viral replication. IFN\u3b3 and CD69 mRNA expression, as well as the percentage of perforin-producing CD8+ T Lymphocytes, were analyzed 3 and 7-days post in vitro HIV-1-infection, respectively. The effect of rhERAP2 addition in cell cultures on T cell apoptosis, proliferation, activation, and maturation was evaluated as well on 24 h-stimulated PBMCs. Results: ERAP2 can be secreted from human MDMs in response to IFN\u3b3/LPS stimulation. Notably, the addition of rhERAP2 to PBMC and CD8-PBMC cultures resulted in the reduction of viral replication, though these differences were statistically significant only in PBMCs (p < 0.05 in both HomoA and HomoB). This protective effect was associated with an increase in IFN\u3b3 and CD69 mRNA expression and in the percentage of perforin-expressing CD107+CD8+ cells. RhERAP2 addition also resulted in an increase in CD8+ activated lymphocyte (CD25+HLA-DRII+) and Effector Memory/Terminally differentiated CD8+ T cells ratio. Conclusions: This is the first report providing evidence for the release of ERAP2 in the secretome of immunocompetent cells. Data herein also indicate that exogenous ERAP2-FL exerts its protective function against HIV-1 infection, even in HomoB subjects who do not genetically produce it. Presumably, this defensive extracellular feature is only partially dependent on immune system modulation

The fitness landscape of the African salmonella typhimurium ST313 strain D23580 reveals unique properties of the pBT1 plasmid

We have used a transposon insertion sequencing (TIS) approach to establish the fitness landscape of the African Salmonella enterica serovar Typhimurium ST313 strain D23580, to complement our previous comparative genomic and functional transcriptomic studies. We used a genome-wide transposon library with insertions every 10 nucleotides to identify genes required for survival and growth in vitro and during infection of murine macrophages. The analysis revealed genomic regions important for fitness under two in vitro growth conditions. Overall, 724 coding genes were required for optimal growth in LB medium, and 851 coding genes were required for growth in SPI-2-inducing minimal medium. These findings were consistent with the essentiality analyses of other S. Typhimurium ST19 and S. Typhi strains. The global mutagenesis approach also identified 60 sRNAs and 413 intergenic regions required for growth in at least one in vitro growth condition. By infecting murine macrophages with the transposon library, we identified 68 genes that were required for intra-macrophage replication but did not impact fitness in vitro. None of these genes were unique to S. Typhimurium D23580, consistent with a high conservation of gene function between S. Typhimurium ST313 and ST19 and suggesting that novel virulence factors are not involved in the interaction of strain D23580 with murine macrophages. We discovered that transposon insertions rarely occurred in many pBT1 plasmid-encoded genes (36), compared with genes carried by the pSLT-BT virulence plasmid and other bacterial plasmids. The key essential protein encoded by pBT1 is a cysteinyl-tRNA synthetase, and our enzymological analysis revealed that the plasmid-encoded CysRSpBT1 had a lower ability to charge tRNA than the chromosomally-encoded CysRSchr enzyme. The presence of aminoacyl-tRNA synthetases in plasmids from a range of Gram-negative and Gram-positive bacteria suggests that plasmid-encoded essential genes are more common than had been appreciated

PARP inhibition by olaparib or gene knockout blocks asthma-like manifestation in mice by modulating CD4+ T cell function

Background: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. Methods: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4+ T cells. C57BL/6J WT or PARP-1-/- mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. Results: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-\u3b3. These traits were associated with a decrease in splenic CD4+ T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1-/- mice. Adoptive transfer of Th2-skewed OT-II-WT CD4+ T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-\u3b3, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1-/-mice suggesting a role for PARP-1 in CD4+ T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4+ T cells while causing a moderate elevation in t-bet and ifn-\u3b3 expression in Th1-skewed CD4+ T cells. Conclusions: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials

Thiazolides elicit anti-viral innate immunity and drastically reduce HIV replication in vitro

Background: Nitazoxanide (Alinia\uae, NTZ) and tizoxanide (TIZ), its active circulating metabolite, belong to a new class of agents known as thiazolides (TZD) that are endowed with a broad anti-infective activity. TIZ and RM-4848, the active circulating metabolite of the new generation thiazolide RM-5038, were also recently shown to potently stimulate innate immunity in vitro. Because natural resistance to HIV-1 infection in HIV-exposed seronegative (HESN) individuals is associated with strong innate immune responses, we verified whether TIZ and RM4848 could reduce in vitro susceptibility to HIV-1. Methods: Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors were infected in vitro with HIV-1BaL in the presence/absence of TIZ or RM-4848. HIV-1 p24 was measured 7 and 10 days post infection. The immunomodulatory abilities of TZD and RM-4848 were evaluated by examining the expression of genes that are part of the type I IFN pathway and are stimulated by IFN (ISGs), as well as the downstream production of cytokines and chemokines. Results: TIZ and RM-4848 drastically (>87%) and consistently inhibited in vitro HIV-1 replication. This was associated with the activation of innate immune responses and with the up-regulation of several ISGs, including those involved in cholesterol metabolism and efflux and in particular cholesterol-25 hydroxylase (CH25H). Conclusions: Thiazolides inhibit HIV-1 replication in vitro possibly as a result of their ability to stimulate potent and multifaceted antiviral immune responses. These data warrant the exploration of thiazolides as preventive/therapeutic agent in HIV infection

The interleukin 21 (IL 21)/ microRNA-29 (miR-29) axis is associated with natural resistance to HIV-1 infection

Background: Interleukin-21 (IL-21) modulates HIV-1 infection through the elicitation of different antiviral mechanisms, including Th17 lineage commitment and induction of microRNA (miR)-29,a mi RNA endowed with anti-HIV activity. As miR-29 expression is significantly increased in HIV-1-exposed seronegative individuals (HESN), we investigated the role of miR-29/IL21 axis in the natural control of HIV-1 infection. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 15 Italian sexually exposed HESN and 15 HIV-unexposed healthy controls were in-vitro infected with an R5-tropic HIV-1(Ba-L) strain. Seven days post HIV-1 infection we evaluated: 1) p24 production (ELISA); 2) CD4(+)/IL-21(+) and CD4(+)/IL-17(+) T lymphocytes (FACS); 3) IL-17 concentration in supernatants (ELISA); and 4) IL-6, IL-17, IL-21, and miR-29a,b,c expression by CD4(+) T lymphocytes as well as perforin and granzyme by peripheral blood mononuclear cells (qPCR). The same analyses were performed on the 15 HIV-positive partners. Results: At baseline IL-6 expression alone was increased in HESN compared to healthy controls. Seven days after in-vitro HIV-1 infection, nevertheless, differences emerged. Thus, CD4(+)/IL21(+) and CD4(+)/IL17(+) T lymphocytes, as well as IL-21 and IL-17 expression and production were significantly augmented in HESN compared to healthy controls. Interestingly, IL-21 upregulation correlated with a significantly increased expression of miR-29a,b,c and a reduced susceptibility to in-vitro HIV-1 infection in HESN alone. No differences were observed in perforin and granzyme expression. Conclusion: The IL-21/miR-29 axis is upregulated by HIV-1 infection in HESN suggesting its involvement in the natural resistance to HIV-1 infection in HESN. Approaches that exogenously increase IL-21 production or prompt preexisting cellular IL-21 reservoir could confine the magnitude of the initial HIV-1 infection

PARP is activated in human asthma and its inhibition by olaparib blocks house dust mite-induced disease in mice

Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-\u3b3 or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice