Anne E. Goldfeld

IL-10–producing and naturally occurring CD4+ Tregs: limiting collateral damage

Effective immune responses against pathogens are sometimes accompanied by strong inflammatory reactions. To minimize damage to self, the activation of the immune system also triggers anti-inflammatory circuits. Both inflammatory and anti-inflammatory reactions are normal components of the same immune response, which coordinately fight infections while preventing immune pathology. IL-10 is an important suppressive cytokine, produced by a large number of immune cells in addition to the antigen-driven IL-10-producing regulatory and the naturally occurring suppressor CD4+ T cells, which is a key player in anti-inflammatory immune responses. However, additional mechanisms have evolved to ensure that pathogen eradication is achieved with minimum damage to the host. Here we discuss those mechanisms that operate to regulate effector immune responses.

Earlier versus Later Start of Antiretroviral Therapy in HIV-Infected Adults with Tuberculosis

BACKGROUND Tuberculosis remains an important cause of death among patients infected with the human immunodeficiency virus (HIV). Robust data are lacking with regard to the timing for the initiation of antiretroviral therapy (ART) in relation to the start of antituberculosis therapy. METHODS We tested the hypothesis that the timing of ART initiation would significantly affect mortality among adults not previously exposed to antiretroviral drugs who had newly diagnosed tuberculosis and CD4+ T-cell counts of 200 per cubic millimeter or lower. After beginning the standard, 6-month treatment for tuberculosis, patients were randomly assigned to either earlier treatment (2 weeks after beginning tuberculosis treatment) or later treatment (8 weeks after) with stavudine, lamivudine, and efavirenz. The primary end point was survival. RESULTS A total of 661 patients were enrolled and were followed for a median of 25 months. The median CD4+ T-cell count was 25 per cubic millimeter, and the median viral load was 5.64 log(10) copies per milliliter. The risk of death was significantly reduced in the group that received ART earlier, with 59 deaths among 332 patients (18%), as compared with 90 deaths among 329 patients (27%) in the later-ART group (hazard ratio, 0.62; 95% confidence interval [CI]; 0.44 to 0.86; P=0.006). The risk of tuberculosis-associated immune reconstitution inflammatory syndrome was significantly increased in the earlier-ART group (hazard ratio, 2.51; 95% CI, 1.78 to 3.59; P<0.001). Irrespective of the study group, the median gain in the CD4+ T-cell count was 114 per cubic millimeter, and the viral load was undetectable at week 50 in 96.5% of the patients. CONCLUSIONS Initiating ART 2 weeks after the start of tuberculosis treatment significantly improved survival among HIV-infected adults with CD4+ T-cell counts of 200 per cubic millimeter or lower. (Funded by the French National Agency for Research on AIDS and Viral Hepatitis and the National Institutes of Health; CAMELIA ClinicalTrials.gov number, NCT01300481.).

IL-10–producing T cells suppress immune responses in anergic tuberculosis patients

The lethality of Mycobacterium tuberculosis remains the highest among infectious organisms and is linked to inadequate immune response of the host. Containment and cure of tuberculosis requires an effective cell-mediated immune response, and the absence, during active tuberculosis infection, of delayed-type hypersensitivity (DTH) responses to mycobacterial antigens, defined as anergy, is associated with poor clinical outcome. To investigate the biochemical events associated with this anergy, we screened 206 patients with pulmonary tuberculosis and identified anergic patients by their lack of dermal reactivity to tuberculin purified protein derivative (PPD). In vitro stimulation of T cells with PPD induced production of IL-10, IFN-gamma, and proliferation in PPD(+) patients, whereas cells from anergic patients produced IL-10 but not IFN-gamma and failed to proliferate in response to this treatment. Moreover, in anergic patients IL-10-producing T cells were constitutively present, and T-cell receptor-mediated (TCR-mediated) stimulation resulted in defective phosphorylation of TCRzeta and defective activation of ZAP-70 and MAPK. These results show that T-cell anergy can be induced by antigen in vivo in the intact human host and provide new insights into mechanisms by which M. tuberculosis escapes immune surveillance.

A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo.

ABSTRACT The tumor necrosis factor alpha (TNF-α) gene is rapidly activated by lipopolysaccharide (LPS). Here, we show that extracellular signal-regulated kinase (ERK) kinase activity but not calcineurin phosphatase activity is required for LPS-stimulated TNF-α gene expression. In LPS-stimulated macrophages, the ERK substrates Ets and Elk-1 bind to the TNF-α promoter in vivo. Strikingly, Ets and Elk-1 bind to two TNF-α nuclear factor of activated T cells (NFAT)-binding sites, which are required for calcineurin and NFAT-dependent TNF-α gene expression in lymphocytes. The transcription factors ATF-2, c-jun, Egr-1, and Sp1 are also inducibly recruited to the TNF-α promoter in vivo, and the binding sites for each of these activators are required for LPS-stimulated TNF-α gene expression. Furthermore, assembly of the LPS-stimulated TNF-α enhancer complex is dependent upon the coactivator proteins CREB binding protein and p300. The finding that a distinct set of transcription factors associates with a fixed set of binding sites on the TNF-α promoter in response to LPS stimulation lends new insights into the mechanisms by which complex patterns of gene regulation are achieved.

Control of HIV-1 viremia and protection from AIDS are associated with HLA-Bw4 homozygosity

Certain HLA-B antigens have been associated with lack of progression to AIDS. HLA-B alleles can be divided into two mutually exclusive groups based on the expression of the molecular epitopes HLA-Bw4 and HLA-Bw6. Notably, in addition to its role in presenting viral peptides for immune recognition, the HLA-Bw4, but not HLA-Bw6, motif functions as a ligand for a natural killer cell inhibitory receptor (KIR). Here, we show that profound suppression of HIV-1 viremia is significantly associated with homozygosity for HLA-B alleles that share the HLA-Bw4 epitope. Furthermore, homozygosity for HLA-Bw4 alleles was also significantly associated with the ability to remain AIDS free and to maintain a normal CD4 T cell count in a second cohort of HIV-1-infected individuals with well defined dates of seroconversion. This association was independent of the presence of a mutation in CC chemokine receptor 5 (CCR5) associated with resistance to HIV-1 infection, and it was independent of the presence of HLA alleles that could potentially confound the results. We conclude that homozygosity for HLA-Bw4-bearing B alleles is associated with a significant advantage and that the HLA-Bw4 motif is important in AIDS pathogenesis.

Stimulus-Specific Assembly of Enhancer Complexes on the Tumor Necrosis Factor Alpha Gene Promoter

ABSTRACT The human tumor necrosis factor alpha (TNF-α) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-α enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-α gene transcription and selectively associates with the TNF-α promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-α transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

Identification of a Macrophage-Specific Chromatin Signature in the IL-10 Locus

The molecular mechanisms that regulate expression of the immunosuppressive cytokine IL-10 remain poorly understood. In this study, by measuring sensitivity to DNase I digestion, we show that production of IL-10 by primary mouse bone marrow-derived macrophages stimulated through pattern recognition receptors was associated with chromatin remodeling of the IL-10 locus. We also demonstrate that the IL-10 locus is remodeled in primary Th2 cells and IL-10-producing regulatory T cells that have been differentiated in vitro. Strikingly, a novel DNase I-hypersensitive site (HSS-4.5) was identified in stimulated macrophages, but not in T cells. We show that hyperacetylated histones were recruited to this site in stimulated macrophages. Furthermore, HSS-4.5 is highly conserved and contains a putative NF-κB binding site. In support of a function for this site, NF-κB p65/RelA was recruited to HSS-4.5 in vivo and its activation was required for optimal IL-10 gene expression in LPS-stimulated macrophages.

T cells and adaptive immunity to Mycobacterium tuberculosis in humans.

The adaptive immune response mediated by T cells is critical for control of Mycobacterium tuberculosis (M. tuberculosis) infection in humans. However, the M. tuberculosis antigens and host T‐cell responses that are required for an effective adaptive immune response to M. tuberculosis infection are yet to be defined. Here, we review recent findings on CD4+ and CD8+ T‐cell responses to M. tuberculosis infection and examine the roles of distinct M. tuberculosis‐specific T‐cell subsets in control of de novo and latent M. tuberculosis infection, and in the evolution of T‐cell immunity to M. tuberculosis in response to tuberculosis treatment. In addition, we discuss recent studies that elucidate aspects of M. tuberculosis‐specific adaptive immunity during human immunodeficiency virus co‐infection and summarize recent findings from vaccine trials that provide insight into effective adaptive immune responses to M. tuberculosis infection.

Engagement of tumor necrosis factor (TNF) receptor 1 leads to ATF-2- and p38 mitogen-activated protein kinase-dependent TNF-alpha gene expression.

Engagement of the tumor necrosis factor-α (TNF-α) receptors by the TNF-α ligand results in the rapid induction of TNF-α gene expression. The study presented here shows that autoregulation of TNF-α gene transcription by selective signaling through tumor necrosis factor receptor 1 (TNFR1) requires p38 mitogen-activated protein (MAP) kinase activity and the binding of the transcription factors ATF-2 and Jun to the TNF-α cAMP-response element (CRE) promoter element. Consistent with these findings, TNFR1 engagement results in increased p38 MAP kinase activity and p38-dependent phosphorylation of ATF-2. Furthermore, overexpression of MADD (MAP kinase-activatingdeath domain protein), an adapter protein that binds to the death domain of TNFR1 and activates MAP kinase cascades, results in CRE-dependent induction of TNF-α gene expression. Thus, the TNF-α CRE site is the target of TNFR1 stimulation and mediates the autoregulation of TNF-α gene transcription.

Antigen-specific and persistent tuberculin anergy in a cohort of pulmonary tuberculosis patients from rural Cambodia

Purified protein derivative (PPD) skin testing is used to identify persons infected with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immune responses to Mtb. However, lack of skin induration to intradermal injection of PPD or PPD anergy is observed in a subset of patients with active tuberculosis (TB). To investigate the sensitivity and persistence of PPD reactivity and its in vitro correlates during active TB disease and after successful chemotherapy, we evaluated the distribution of skin size induration after intradermal injection of PPD among 364 pulmonary TB patients in Cambodia. A subset of 25 pulmonary TB patients who had a positive skin reaction to mumps and/or candida antigens showed persistent anergy to PPD after successful completion of TB therapy. Strikingly, in vitro stimulation of T cells from persistently anergic TB patients with mumps but not PPD resulted in T cell proliferation, and lower levels of IL-2 and IFN-γ and higher levels of IL-10 were detected in PPD-stimulated cellular cultures from PPD-anergic as compared with PPD-reactive pulmonary TB patients. These results show that anergy to PPD is antigen-specific and persistent in a subset of immunocompetent pulmonary TB patients and is characterized by antigen-specific impaired T cell proliferative responses and a distinct pattern of cytokine production including reduced levels of IL-2.