Cynthia Kanagaratham

Distinct Tryptophan Catabolism and Th17/Treg Balance in HIV Progressors and Elite Controllers

Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohort of HIV-infected patients with different clinical outcomes: ART-naïve, Successfully Treated (ST), and elite controllers (EC). In ART-naïve patients, increased IDO activity/expression, together with elevated levels of TNF-α and sCD40L, were associated with Treg expansion and an altered Th17/Treg balance. These alterations were normalized under ART. In contrast, Trp 2,3-dioxegenase (TDO) expression was dramatically lower in EC when compared to all other groups. Interestingly, EC displayed a distinctive Trp metabolism characterized by low Trp plasma levels similar to ART-naïve patients without accumulating immunosuppressive Kyn levels which was accompanied by a preserved Th17/Treg balance. These results suggest a distinctive Trp catabolism and Th17/Treg balance in HIV progressors and EC. Thus, IDO-induced immune-metabolism may be considered as a new inflammation-related marker for HIV-1 disease progression.

Immunosuppressive tryptophan catabolism and gut mucosal dysfunction following early HIV infection

BACKGROUND Tryptophan (Trp) catabolism into kynurenine (Kyn) contributes to immune dysfunction in chronic human immunodeficiency virus (HIV) infection. To better define the relationship between Trp catabolism, inflammation, gut mucosal dysfunction, and the role of early antiretroviral therapy (ART), we prospectively assessed patients early after they acquired HIV. METHODS Forty patients in the early phase of infection were longitudinally followed for 12 months after receiving a diagnosis of HIV infection; 24 were untreated, and 16 were receiving ART. Kyn/Trp ratio, regulatory T-cells (Tregs) frequency, T-cell activation, dendritic cell counts, and plasma levels of gut mucosal dysfunction markers intestinal-type fatty acid-binding protein, soluble suppression of tumorigenicity 2, and lipopolysaccharide were assessed. RESULTS Compared with healthy subjects, patients in the early phase of infection presented with elevated Kyn/Trp ratios, which further increased in untreated patients but normalized in ART recipients. Accordingly, in untreated subjects, the elevated Treg frequency observed at baseline continued to increase over time. The highest CD8(+) T-cell activation was observed during the early phase of infection and decreased in untreated patients, whereas activation normalized in ART recipients. The Kyn/Trp ratio was positively associated with CD8(+) T-cell activation and levels of inflammatory cytokines (interleukin 6, interferon γ-inducible protein 10, interleukin 18, and tumor necrosis factor α) and negatively associated with dendritic cell frequencies at baseline and in untreated patients. However, ART did not normalize plasma levels of gut mucosal dysfunction markers. CONCLUSIONS Early initiation of ART normalized enhanced Trp catabolism and immune activation but did not improve plasma levels of gut mucosal dysfunction markers.

Secretory Leukocyte Protease Inhibitor Plays an Important Role in the Regulation of Allergic Asthma in Mice

Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.

Soluble CD40-ligand ( sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection

CD40/CD40‐ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO‐induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV‐infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti‐retroviral therapy (ART)‐naive, ART‐successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme‐linked immunosorbent assay (ELISA). IDO‐mRNA expression was quantified by reverse transcription–polymerase chain reaction (RT–PCR). The in‐vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART‐naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART‐naive alone, sCD40L was correlated with T cell activation, IDO‐mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV‐infected patients. In‐vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co‐expression of CD38/human leucocyte antigen D‐related (HLA‐DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO‐induced Trp catabolism and Treg expansion.

Nucleolar targeting of the chaperone HSC70 is regulated by stress, cell signaling and a composite targeting signal which is controlled by autoinhibition

Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.

Assessment of chloroquine as a modulator of immune activation to improve CD4 recovery in immune nonresponding HIV‐infected patients receiving antiretroviral therapy

Chloroquine (CQ), an anti‐inflammatory drug, inhibits Toll‐like receptor (TLR) signalling in plasmacytoid dendritic cells (pDCs) and may be beneficial for HIV‐infected patients in whom immune activation persists despite effective antiretroviral therapy (ART). The effect of CQ on CD4 T‐cell recovery and immune activation in immune nonresponding patients receiving successful ART was therefore studied.

Polymorphisms in TOLL-Like Receptor Genes and their Roles in Allergic Asthma and Atopy

Allergic asthma is a chronic inflammatory disease of the lung airways cause by genetic and environmental factors. Two quantifiable phenotypes of this disease are airway hyperresponsiveness and atopy. TOLL-like receptors (TLRs) are a family of intracellular and cell surface receptors that can respond to pathogen associated molecular patterns involved in the pathogenesis of asthma. Macrophages, one of the main immune cells involved in asthma, express a variety of TLRs, including TLR 2, 4, 5, 6, 7, 8 and 9. This review focuses on polymorphisms found in TLR genes expressed in macrophages, and their role in asthma. Human studies have detected polymorphisms in TLR genes associated with asthma phenotypes, and studies using murine models have shown that some receptors and their agonistic or antagonistic ligands are capable of modulating the cytokine profile in asthmatics in a protective manner. Therefore, certain receptors and their ligands are being explored as potential immunotherapies for asthma. Recently, several patents have been filed protecting inventions for treating asthma through the use of TLRs and their ligands.

Gene-Metabolite Expression in Blood Can Discriminate Allergen-Induced Isolated Early from Dual Asthmatic Responses

Some asthmatic individuals undergoing allergen inhalation challenge develop an isolated early response whereas others develop a dual response (early plus late response). In the present study we have used transcriptomics (microarrays) and metabolomics (mass spectrometry) of peripheral blood to identify molecular patterns that can discriminate allergen-induced isolated early from dual asthmatic responses. Peripheral blood was obtained prior to (pre-) and 2 hours post allergen inhalation challenge from 33 study participants. In an initial cohort of 14 participants, complete blood counts indicated significant differences in neutrophil and lymphocyte counts at pre-challenge between early and dual responders. At post-challenge, significant genes (ALOX15, FADS2 and LPCAT2) and metabolites (lysolipids) were enriched in lipid metabolism pathways. Enzymes encoding for these genes are involved in membrane biogenesis and metabolism of fatty acids into pro-inflammatory and anti-inflammatory mediators. Correlation analysis indicated a strong negative correlation between ALOX15, FADS2, and IL5RA expression with 2-arachidonoylglycerophosphocholine levels in dual responders. However, measuring arachidonic acid and docosahexaenoic acid levels in a validation cohort of 19 participants indicated that the free form of DHA (nmoles/µg of protein) was significantly (p = 0.03) different between early and dual responders after allergen challenge. Collectively these results may suggest an imbalance in lipid metabolism which dictates pro- (anti-) inflammatory and pro-resolving mechanisms. Future studies with larger sample sizes may reveal novel mechanisms and therapeutic targets of the late phase asthmatic response.

Promoter Deletion Analysis Using a Dual-Luciferase Reporter System

Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.

Fenretinide Prevents Inflammation and Airway Hyperresponsiveness in a Mouse Model of Allergic Asthma

Arachidonic acid (AA) and docosahexaenoic acid (DHA) play important roles in inflammation and disease progression, where AA is viewed as proinflammatory and DHA as antiinflammatory. We observe in our model of allergic asthma that the AA/DHA ratio is significantly skewed in a proinflammatory direction. Fenretinide, a vitamin A derivative, has been shown to correct fatty acid imbalances in other diseases. Therefore, we explored if fenretinide can have a protective effect in allergic asthma. To accomplish this, we measured the levels of AA and DHA in the lungs of nonallergic, ovalbumin-induced allergic, and fenretinide-treated allergic mice. We also investigated the effect of allergic asthma and fenretinide treatment on markers of oxidative stress, levels of metabolites, IgE production, airway hyperresponsiveness, and histological changes. Our data demonstrate that treatment of allergen-sensitized mice with fenretinide before allergen challenge prevents ovalbumin-induced changes in the AA/DHA ratio. The levels of several metabolites, such as serotonin, and markers of cellular stress, which are increased after ovalbumin challenge, are also controlled by fenretinide treatment. We observed the protective effect of fenretinide against ovalbumin-induced airway hyperresponsiveness and inflammation in the lungs, illustrated by a complete block in the infiltration of inflammatory cells to the airways and dramatically diminished goblet cell proliferation, even though IgE remained high. Our results demonstrate that fenretinide is an effective agent targeting inflammation, oxidation, and lung pathology observed in allergic asthma.