Epidermal Growth Factor Receptor-Responsive Indoleamine 2,3-Dioxygenase Confers Immune Homeostasis During Shigella flexneri Infection.

The resolution of Shigella flexneri infection-associated hyperinflammation is crucial for host survival. Using in vitro and in vivo models of shigellosis, we found that S. flexneri induces the expression of indoleamine 2,3-dioxygenase 1 (IDO1) through the nucleotide oligomerization domain 2 (NOD2) and epidermal growth factor receptor (EGFR) signaling pathway. Congruently, abrogation of NOD2 or EGFR compromises the ability of S. flexneri to induce IDO1 expression. We observed that the loss of IDO1 function in vivo exacerbates shigellosis by skewing the inflammatory cytokine response, disrupting colon epithelial barrier integrity and consequently limiting the host life-span. Interestingly, administration of recombinant EGF rescued mice from IDO1 inhibition-driven aggravated shigellosis by restoring the cytokine balance and subsequently restricting bacterial growth. This is the first study that underscores the direct implication of the NOD2-EGFR axis in IDO1 production and its crucial homeostatic contributions during shigellosis. Together, these findings reveal EGF as a potential therapeutic intervention for infectious diseases.

Aspergillus fumigatus Cell Wall α-(1,3)-Glucan Stimulates Regulatory T-Cell Polarization by Inducing PD-L1 Expression on Human Dendritic Cells.

Background: Human dendritic cell (DC) response to α-(1,3)-glucan polysaccharide of Aspergillus fumigatus and ensuing CD4+ T-cell polarization are poorly characterized. Methods: α-(1,3)-Glucan was isolated from A. fumigatus conidia and mycelia cell wall. For the analysis of polarization, DCs and autologous naive CD4+ T cells were cocultured. Phenotype of immune cells was analyzed by flow cytometry, and cytokines by enzyme-linked immunosorbent assay (ELISA). Blocking antibodies were used to dissect the role of Toll-like receptor 2 (TLR2) and programmed death-ligand 1 (PD-L1) in regulating α-(1,3)-glucan-mediated DC activation and T-cell responses. DCs from TLR2-deficient mice were additionally used to consolidate the findings. Results: α-(1,3)-Glucan induced the maturation of DCs and was dependent in part on TLR2. "α-(1,3)-Glucan-educated" DCs stimulated the activation of naive T cells and polarized a subset of these cells into CD4+CD25+FoxP3+ regulatory T cells (Tregs). Mechanistically, Treg stimulation by α-(1,3)-glucan was dependent on the PD-L1 pathway that negatively regulated interferon-gamma (IFN-γ) secretion. Short α-(1,3)-oligosaccharides lacked the capacity to induce maturation of DCs but significantly blocked α-(1,3)-glucan-induced Treg polarization. Conclusions: PD-L1 dictates the balance between Treg and IFN-γ responses induced by α-(1,3)-glucan. Our data provide a rationale for the exploitation of immunotherapeutic approaches that target PD-1-PD-L1 to enhance protective immune responses to A. fumigatus infections.

Regulatory T cell frequency, but not plasma IL-33 levels, represents potential immunological biomarker to predict clinical response to intravenous immunoglobulin therapy.

BACKGROUND: Intravenous immunoglobulin (IVIG) is a polyspecific pooled immunoglobulin G preparation and one of the commonly used therapeutics for autoimmune diseases including those of neurological origin. A recent report in murine model proposed that IVIG expands regulatory T (Treg) cells via induction of interleukin 33 (IL-33). However, translational insight on these observations is lacking. METHODS: Ten newly diagnosed Guillain-Barré syndrome (GBS) patients were treated with IVIG at the rate of 0.4 g/kg for three to five consecutive days. Clinical evaluation for muscular weakness was performed by Medical Research Council (MRC) and modified Rankin scoring (MRS) system. Heparinized blood samples were collected before and 1, 2, and 4-5 weeks post-IVIG therapy. Peripheral blood mononuclear cells were stained for surface CD4 and intracellular Foxp3, IFN-γ, and tumor necrosis factor alpha (TNF-α) and were analyzed by flow cytometry. IL-33 and prostaglandin E2 in the plasma were measured by ELISA. RESULTS: The fold changes in plasma IL-33 at week 1 showed no correlation with the MRC and MRS scores at weeks 1, 2, and ≥4 post-IVIG therapy. Clinical recovery following IVIG therapy appears to be associated with Treg cell response. Contrary to murine study, there was no association between the fold changes in IL-33 at week 1 and Treg cell frequency at weeks 1, 2, and ≥4 post-IVIG therapy. Treg cell-mediated clinical response to IVIG therapy in GBS patients was associated with reciprocal regulation of effector T cells-expressing TNF-α. CONCLUSION: Treg cell expansion by IVIG in patients with autoimmune diseases lack correlation with IL-33. Treg cell frequency, but not plasma IL-33 levels, represents potential immunological biomarker to predict clinical response to IVIG therapy.

Intravenous immunoglobulin expands regulatory T cells via induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) play a critical role in the maintenance of immune tolerance. Intravenous immunoglobulin (IVIg), a therapeutic preparation of normal pooled human IgG, expands Tregs in various experimental models and in patients. However, the cellular and molecular mechanisms by which IVIg expands Tregs are relatively unknown. As Treg expansion in the periphery requires signaling by antigen-presenting cells such as dendritic cells (DCs) and IVIg has been demonstrated to modulate DC functions, we hypothesized that IVIg induces distinct signaling events in DCs that subsequently mediate Treg expansion. We demonstrate that IVIg expands Tregs via induction of cyclooxygenase (COX)-2-dependent prostaglandin E2 (PGE2) in human DCs. However, costimulatory molecules of DCs such as programmed death ligands, OX40 ligand, and inducible T-cell costimulator ligands were not implicated. Inhibition of PGE2 synthesis by COX-2 inhibitors prevented IVIg-mediated Treg expansion in vitro and significantly diminished IVIg-mediated Treg expansion in vivo and protection from disease in experimental autoimmune encephalomyelitis model. IVIg-mediated COX-2 expression, PGE2 production, and Treg expansion were mediated in part via interaction of IVIg and F(ab')2 fragments of IVIg with DC-specific intercellular adhesion molecule-3-grabbing nonintegrin. Our results thus uncover novel cellular and molecular mechanism by which IVIg expands Tregs.

Alkyl chain substituted 1,9-pyrazoloanthrones exhibit prominent inhibitory effect on c-Jun N-terminal kinase (JNK).

N-Alkyl substituted pyrazoloanthrone derivatives were synthesized, characterized and tested for their in vitro inhibitory activity over c-Jun N-terminal kinase (JNK). Among the tested molecules, a few derivatives showed significant inhibitory activity against JNK with minimal off-target effect on other mitogen-activated protein kinase (MAP kinase) family members such as MEK1/2 and MKK3,6. These results suggested that N-alkyl (propyl and butyl) bearing pyrazoloanthrone scaffolds provide promising therapeutic inhibitors for JNK in regulating inflammation associated disorders.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras.

BACKGROUND: A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. METHODS: Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). RESULTS: M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. CONCLUSIONS: The pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

Intracellular pathogen sensor NOD2 programs macrophages to trigger Notch1 activation.

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-α or IFN-γ-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

Pattern recognition and cellular immune responses to novel Mycobacterium tuberculosis-antigens in individuals from Belarus.

BACKGROUND: Tuberculosis (TB) is an enduring health problem worldwide and the emerging threat of multidrug resistant (MDR) TB and extensively drug resistant (XDR) TB is of particular concern. A better understanding of biomarkers associated with TB will aid to guide the development of better targets for TB diagnosis and for the development of improved TB vaccines. METHODS: Recombinant proteins (n = 7) and peptide pools (n = 14) from M. tuberculosis (M.tb) antigens associated with M.tb pathogenicity, modification of cell lipids or cellular metabolism, were used to compare T cell immune responses defined by IFN-γ production using a whole blood assay (WBA) from i) patients with TB, ii) individuals recovered from TB and iii) individuals exposed to TB without evidence of clinical TB infection from Minsk, Belarus. RESULTS: We identified differences in M.tb target peptide recognition between the test groups, i.e. a frequent recognition of antigens associated with lipid metabolism, e.g. cyclopropane fatty acyl phospholipid synthase. The pattern of peptide recognition was broader in blood from healthy individuals and those recovered from TB as compared to individuals suffering from pulmonary TB. Detection of biologically relevant M.tb targets was confirmed by staining for intracellular cytokines (IL-2, TNF-α and IFN-γ) in T cells from non-human primates (NHPs) after BCG vaccination. CONCLUSIONS: PBMCs from healthy individuals and those recovered from TB recognized a broader spectrum of M.tb antigens as compared to patients with TB. The nature of the pattern recognition of a broad panel of M.tb antigens will devise better strategies to identify improved diagnostics gauging previous exposure to M.tb; it may also guide the development of improved TB-vaccines.

Nitric oxide is involved in Mycobacterium bovis bacillus Calmette-Guérin-activated Jagged1 and Notch1 signaling.

Pathogenic mycobacteria have evolved unique strategies to survive within the hostile environment of macrophages. Modulation of key signaling cascades by NO, generated by the host during infection, assumes critical importance in overall cell-fate decisions. We show that NO is a critical factor in Mycobacterium bovis bacillus Calmette-Guérin-mediated Notch1 activation, as the generation of activated Notch1 or expression of Notch1 target genes matrix metalloproteinase-9 (MMP-9) or Hes1 was abrogated in macrophages derived from inducible NO synthase (iNOS) knockout (iNOS(-/-)), but not from wild-type, mice. Interestingly, expression of the Notch1 ligand Jagged1 was compromised in M. bovis bacillus Calmette-Guérin-stimulated iNOS(-/-) macrophages, and loss of Jagged1 expression or Notch1 signaling could be rescued by NO donors. Signaling perturbations or genetic approaches implicated that robust expression of MMP-9 or Hes1 required synergy and cross talk between TLR2 and canonical Notch1-PI3K cascade. Further, CSL/RBP-Jk contributed to TLR2-mediated expression of MMP-9 or Hes1. Correlative evidence shows that, in a murine model for CNS tuberculosis, this mechanism operates in vivo only in brains derived from WT but not from iNOS(-/-) mice. Importantly, we demonstrate the activation of Notch1 signaling in vivo in granulomatous lesions in the brains of Mycobacterium tuberculosis-infected human patients with tuberculous meningitis. Current investigation identifies NO as a pathological link that modulates direct cooperation of TLR2 with Notch1-PI3K signaling or Jagged1 to regulate specific components of TLR2 responses. These findings provide new insights into mechanisms by which Notch1, TLR2, and NO signals are integrated in a cross talk that modulates a defined set of effector functions in macrophages.

PE_PGRS antigens of Mycobacterium tuberculosis induce maturation and activation of human dendritic cells.

Mycobacterium tuberculosis, the causative agent of pulmonary tuberculosis, infects one-third of the world's population. Activation of host immune responses for containment of mycobacterial infections involves participation of innate immune cells, such as dendritic cells (DCs). DCs are sentinels of the immune system and are important for eliciting both primary and secondary immune responses to pathogens. In this context, to understand the molecular pathogenesis of tuberculosis and host response to mycobacteria and to conceive prospective vaccine candidates, it is important to understand how cell wall Ags of M. tuberculosis and, in particular, the proline-glutamic acid_polymorphic guanine-cytosine-rich sequence (PE_PGRS) family of proteins modulate DC maturation and function. In this study, we demonstrate that two cell wall-associated/secretory PE_PGRS proteins, PE_PGRS 17 (Rv0978c) and PE_PGRS 11 (Rv0754), recognize TLR2, induce maturation and activation of human DCs, and enhance the ability of DCs to stimulate CD4(+) T cells. We further found that PE_PGRS protein-mediated activation of DCs involves participation of ERK1/2, p38 MAPK, and NF-kappaB signaling pathways. Priming of human DCs with IFN-gamma further augmented PE_PGRS 17 or PE_PGRS 11 Ag-induced DC maturation and secretion of key proinflammatory cytokines. Our results suggest that by activating DCs, PE_PGRS proteins, important mycobacterial cell wall Ags, could potentially contribute in the initiation of innate immune responses during tuberculosis infection and hence regulate the clinical course of tuberculosis.

The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress.

Mycobacterium tuberculosis utilizes unique strategies to survive amid the hostile environment of infected host cells. Infection-specific expression of a unique mycobacterial cell surface antigen that could modulate key signaling cascades can act as a key survival strategy in curtailing host effector responses like oxidative stress. We demonstrate here that hypothetical PE_PGRS11 ORF encodes a functional phosphoglycerate mutase. The transcriptional analysis revealed that PE_PGRS11 is a hypoxia-responsive gene, and enforced expression of PE_PGRS11 by recombinant adenovirus or Mycobacterium smegmatis imparted resistance to alveolar epithelial cells against oxidative stress. PE_PGRS11-induced resistance to oxidative stress necessitated the modulation of genetic signatures like induced expression of Bcl2 or COX-2. This modulation of specific antiapoptotic molecular signatures involved recognition of PE_PGRS11 by TLR2 and subsequent activation of the PI3K-ERK1/2-NF-κB signaling axis. Furthermore, PE_PGRS11 markedly diminished H(2)O(2)-induced p38 MAPK activation. Interestingly, PE_PGRS11 protein was exposed at the mycobacterial cell surface and was involved in survival of mycobacteria under oxidative stress. Furthermore, PE_PGRS11 displayed differential B cell responses during tuberculosis infection. Taken together, our investigation identified PE_PGRS11 as an in vivo expressed immunodominant antigen that plays a crucial role in modulating cellular life span restrictions imposed during oxidative stress by triggering TLR2-dependent expression of COX-2 and Bcl2. These observations clearly provide a mechanistic basis for the rescue of pathogenic Mycobacterium-infected lung epithelial cells from oxidative stress.

Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

Functional role of the PE domain and immunogenicity of the Mycobacterium tuberculosis triacylglycerol hydrolase LipY.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(deltaPE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY(mar), in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY(mar) in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY(mar) was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(deltaPE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(deltaPE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(deltaPE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis.

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.