Adenosine deaminase acting on RNA-1 (ADAR1) inhibits HIV-1 replication in human alveolar macrophages.

While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages.

Differential role for CD80 and CD86 in the regulation of the innate immune response in murine polymicrobial sepsis.

BACKGROUND: Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86(-/-) mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis. METHODOLOGY/PRINCIPAL FINDINGS: CD80(-/-) mice had improved survival after CLP when compared to WT or CD86(-/-) mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80(-/-) mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-kappaB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model. CONCLUSIONS: In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.

CD40 and CD80/86 act synergistically to regulate inflammation and mortality in polymicrobial sepsis.

RATIONALE: Costimulatory molecules, including the CD40-CD154 and CD80/86-CD28 dyads, play a prominent role in regulating inflammation in the adaptive immune response. Studies from our group and others suggest a potentially important role for these costimulatory cascades in innate immunity as well. OBJECTIVES: To determine the role of CD80/86 alone and in combination with CD40 in lethal polymicrobial sepsis in mice and humans. METHODS: The murine cecal ligation and puncture (CLP) model was used to determine the role of CD80/86 alone and in combination with CD40 using wild-type mice, CD80/86(-/-) mice, and novel CD40/80/86(-/-) mice. Expression of cell-bound and soluble costimulatory molecules was assessed in humans via ELISA and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Lethal CLP was associated with up-regulation of CD40 and CD80/86 and their respective ligands CD28 and CD154 on innate effector cells. Blockade or deletion of CD80/86 attenuated mortality and inflammatory cytokine production during CLP. CD40/80/86(-/-) mice exhibited further reductions in mortality, lung injury, and inflammatory cytokine production compared with CD80/86(-/-) mice. Finally, humans with sepsis had increased monocyte expression of CD40 and CD80 compared with healthy control subjects; with higher levels in subjects requiring vasopressor support. Levels of soluble CD28 and CD154 were significantly higher in patients who died compared with those who lived. CONCLUSIONS: These data demonstrate a central role for CD40 and CD80/86 in the innate immune response and suggest that combined inhibition of CD40 and CD80/86 may improve mortality in sepsis. Expression of costimulatory molecules may serve as biomarkers for outcome in septic patients.

Integration of HIV-1 caused STAT3-associated B cell lymphoma in an AIDS patient.

Signal transducer and activator of transcription 3 (STAT3) is a DNA-binding transcription factor activated by multiple cytokines and interferons. High expression of STAT3 has also been implicated in cancer and lymphoma. Here, we show a case of B cell lymphoma in which a defective human immunodeficiency virus 1 (HIV-1) integrated upstream of the first STAT3 coding exon. The lymphoma cells with anaplastic large cell morphology formed multiple nodular lesions in the lung of an acquired immunodeficiency syndrome (AIDS) patient with Kaposi's sarcoma. The provirus had a 5' long terminal repeat (LTR) deletion, but the 3' LTR had stronger promoter activity than the STAT3 promoter in reporter assays. Immunohistochemistry showed increased expression of STAT3 in the nuclei of lymphoma cells. Transfection of STAT3 resulted in transient cell proliferation in primary B cells in vitro. Although this is a very rare case of HIV-1-integrated lymphoma, these data suggest that up-regulation of STAT3 caused by HIV-1 integration resulted in the development of B cell lymphoma in this special case.

Exogenous interferon-alpha and interferon-gamma increase lethality of murine inhalational anthrax.

BACKGROUND: Bacillus anthracis, the etiologic agent of inhalational anthrax, is a facultative intracellular pathogen. Despite appropriate antimicrobial therapy, the mortality from inhalational anthrax approaches 45%, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Type I and Type II Interferons (IFN) are prominent members of the host innate immune response and are required for control of intracellular pathogens. We have previously described a protective role for exogenous Type I and Type II IFNs in attenuating intracellular B.anthracis germination and macrophage cell death in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: We sought to extend these findings in an in vivo model of inhalational anthrax, utilizing the Sterne strain (34F2) of B.anthracis. Mice devoid of STAT1, a component of IFN-alpha and IFN-gamma signaling, had a trend towards increased mortality, bacterial germination and extrapulmonary spread of B.anthracis at 24 hrs. This was associated with impaired IL-6, IL-10 and IL-12 production. However, administration of exogenous IFN-gamma, and to a lesser extent IFN-alpha, at the time of infection, markedly increased lethality. While IFNs were able to reduce the fraction of germinated spores within the lung, they increased both the local and systemic inflammatory response manifest by increases in IL-12 and reductions in IL-10. This was associated with an increase in extrapulmonary dissemination. The mechanism of IFN mediated inflammation appears to be in part due to STAT1 independent signaling. CONCLUSIONS: In conclusion, while endogenous IFNs are essential for control of B.anthracis germination and lethality, administration of exogenous IFNs appear to increase the local inflammatory response, thereby increasing mortality.

Mechanisms of polymorphonuclear neutrophil-mediated induction of HIV-1 replication in macrophages during pulmonary tuberculosis.

BACKGROUND: Pulmonary tuberculosis (TB) can present with polymorphonuclear neutrophil (PMN)-predominant alveolitis. TB accelerates acquired immunodeficiency syndrome by increasing human immunodeficiency virus type 1 (HIV-1) replication and mutation in alveolar macrophages. A 16-kDa CCAAAT/enhancer-binding protein beta (C/EBP beta ) isoform is a strong transcriptional repressor of the HIV long terminal repeat (LTR) in resting alveolar macrophages, leading to latent viral infection; its expression is lost during TB, derepressing the HIV LTR. METHODS: Lung segments were sampled from HIV/Mycobacterium tuberculosis-coinfected patients by means of bronchoalveolar lavage. In vitro coculture experiments defined the mechanism of induction of HIV-1 infection in macrophages by PMNs. RESULTS: Lung segments from patients with PMN-predominant TB had a markedly elevated viral load. Direct contact between activated PMNs and macrophages stimulated HIV-1 replication and LTR transcription and down-regulated inhibitory C/EBP beta . Isolated PMN membranes substituted for PMN contact, derepressing the HIV-1 LTR. The lipid raft fraction of PMN membranes expressed CD40 ligand (CD40L), CD28, and leukocyte function-associated antigen 1 (LFA-1 [i.e., CD11a and CD18]), and PMN activation increased lipid raft expression of CD40L and CD28. Blocking antibodies to CD40L, CD28, and LFA-1 inhibited PMN membrane-mediated HIV-1 LTR derepression. Alternately, cross-linking of macrophage receptors for CD40L, CD28, and LFA-1 (CD40, CD80/86, and intercellular adhesion molecule 1) abolished inhibitory C/EBP beta expression. CONCLUSION: PMN-macrophage contact derepresses the HIV-1 LTR and enhances HIV-1 replication in alveolar macrophages during pulmonary TB. Derepression is mediated through costimulatory molecule signaling.

Interleukin-10 induces inhibitory C/EBPbeta through STAT-3 and represses HIV-1 transcription in macrophages.

Pulmonary tuberculosis (TB) has been characterized by inflammation with increased pro- or anti-inflammatory cytokines produced by macrophages. We have reported that IFN produces inhibitory C/EBPbeta and represses transcription of the HIV-1 LTR in macrophages. STAT-1 and type I IFN receptor knockout mice have macrophages that are defective in IFN signaling, yet LPS stimulation induces inhibitory C/EBPbeta, demonstrating that other cytokines can induce this repressor. LPS or Mycobacterium tuberculosis-derived lipoarabinomannan induce the anti-inflammatory cytokine interleukin (IL)-10, which represses the HIV-1 LTR in differentiated THP-1 macrophages by inducing inhibitory C/EBPbeta. In contrast, in undifferentiated THP-1 monocytes, IL-10 did not inhibit HIV-1 replication or induce C/EBPbeta. IL-10 signal transduction uses STAT-3, and macrophages from STAT-3-/- mice fail to produce inhibitory C/EBPbeta after LPS or IL-10 stimulation. Transfection of STAT-3 into THP-1 cells enhances C/EBPbeta promoter activity. THP-1 differentiation also increases STAT-3 protein, but not STAT-3 gene transcription, and induces a translational regulator, CUG-binding protein, that was essential for production of C/EBPbeta. Differentiation induced post-transcriptional regulation is required to produce inhibitory C/EBPbeta in response to IL-10. Only macrophages are able to repress HIV-1 LTR promoter activity and inhibit viral replication in response to IL-10 or type I IFN.

Mycobacterium tuberculosis-induced CXCR4 and chemokine expression leads to preferential X4 HIV-1 replication in human macrophages.

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1beta), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1alpha). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4-using [corrected] HIV-1, but not of CCR5-using [corrected] HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.

Exogenous gamma and alpha/beta interferon rescues human macrophages from cell death induced by Bacillus anthracis.

During the recent bioterrorism-related outbreaks, inhalational anthrax had a 45% mortality in spite of appropriate antimicrobial therapy, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Alveolar macrophages are likely the first immune cells exposed to inhalational anthrax, and the interferon (IFN) response of these cells comprises an important arm of the host innate immune response to intracellular infection with Bacillus anthracis. Furthermore, IFNs have been used as immunoadjuvants for treatment of another intracellular pathogen, Mycobacterium tuberculosis. We established a model of B. anthracis infection with the Sterne strain (34F(2)) which contains lethal toxin (LeTx). 34F(2) was lethal to murine and human macrophages. Treatment with IFNs significantly improved cell viability and reduced the number of germinated intracellular spores. Infection with 34F(2) failed to induce the latent transcription factors signal transducer and activators of transcription 1 (STAT1) and ISGF-3, which are central to the IFN response. Furthermore, 34F(2) reduced STAT1 activation in response to exogenous alpha/beta IFN, suggesting direct inhibition of IFN signaling. Even though 34F(2) has LeTx, there was no mitogen-activated protein kinase kinase 3 cleavage and p38 was normally induced, suggesting that these early effects of B. anthracis infection in macrophages are independent of LeTx. These data suggest an important role for both IFNs in the control of B. anthracis and the potential benefit of using exogenous IFN as an immunoadjuvant therapy.

Regulation of binding of lamin B receptor to chromatin by SR protein kinase and cdc2 kinase in Xenopus egg extracts.

Participation of multiple kinases in regulation of the binding of lamin B receptor (LBR) to chromatin was suggested previously (Takano, M., Takeuchi, M., Ito, H., Furukawa, K., Sugimoto, K., Omata, S., and Horigome, T. (2002) Eur. J. Biochem. 269, 943-953). To identify these kinases, regulation of the binding of the nucleoplasmic region (NK, amino acid residues 1-211) of LBR to sperm chromatin was studied using a cell cycle-dependent Xenopus egg extract in vitro. The binding was stimulated on specific phosphorylation of the NK fragment by an S-phase egg extract. Protein depletion with beads bearing SF2/ASF, which binds SR protein kinases, abolished this stimulation, suggesting that an SR protein kinase(s) is responsible for the activation of LBR. This was confirmed by direct phosphorylation and activation with recombinant SR protein-specific kinase 1. The binding of the NK fragment to chromatin pretreated with an S-phase extract was suppressed by incubation with an M-phase extract. Enzyme inhibitor experiments revealed that multiple kinases participate in the suppression. One of these kinases was shown to be cdc2 kinase using a specific inhibitor, roscovitine, and protein depletion with beads bearing p13, which specifically binds cdc2 kinase. Experiments involving a mutant NK fragment showed that the phosphorylation of serine 71 by cdc2 kinase is responsible for the suppression.

CD40 contributes to lethality in acute sepsis: in vivo role for CD40 in innate immunity.

Sepsis induces an early inflammatory cascade initiated by the innate immune response. This often results in the development of multisystem organ failure. We examined the role of CD40, a costimulatory molecule that is integral in adaptive immunity, by using a murine model of polymicrobial sepsis. CD40 knockout (KO) mice had delayed death and improved survival after cecal ligation and puncture (CLP). In addition, they had less remote organ injury as manifested by reduced pulmonary capillary leakage. The improvements in survival and remote organ dysfunction in CD40 KO mice were associated with reduced interleukin-6 (IL-6) and IL-10 levels in serum and bronchoalveolar lavage fluid compared to the levels in wild-type (WT) controls. Furthermore, in contrast to WT mice, CD40 KO mice had no induction of the Th1 cytokines IL-12 and gamma interferon in serum or lungs after CLP. The alterations in cytokine production in CD40 KO mice were associated with similar changes in transcription factor activity. After CLP, CD40 KO mice had attenuated activation of nuclear factor kappaB and signal transducer and activator of transcription 3 in both the lung and the liver. Finally, WT mice had increased expression of CD40 on their alveolar macrophages. These data highlight the importance of CD40 activation in the innate immune response during polymicrobial sepsis and the subsequent development of remote organ dysfunction.

Maximal HIV-1 replication in alveolar macrophages during tuberculosis requires both lymphocyte contact and cytokines.

HIV-1 replication is markedly upregulated in alveolar macrophages (AM) during pulmonary tuberculosis (TB). This is associated with loss of an inhibitory CCAAT enhancer binding protein beta (C/EBPbeta) transcription factor and activation of nuclear factor (NF)-kappaB. Since the cellular immune response in pulmonary TB requires lymphocyte--macrophage interaction, a model system was developed in which lymphocytes were added to AM. Contact between lymphocytes and AM reduced inhibitory C/EBPbeta, activated NF-kappaB, and enhanced HIV-1 replication. If contact between lymphocytes and macrophages was prevented, inhibitory C/EBPbeta expression was maintained and the HIV-1 long terminal repeat (LTR) was not maximally stimulated although NF-kappaB was activated. Antibodies that cross-linked macrophage expressed B-7, and vascular cell adhesion molecule and CD40 were used to mimic lymphocyte contact. All three cross-linking antibodies were required to abolish inhibitory C/EBPbeta expression. However, the HIV-1 LTR was not maximally stimulated and NF-kappaB was not activated. Maximal HIV-1--LTR stimulation required both lymphocyte-derived soluble factors, and cross-linking of macrophage expressed costimulatory molecules. High level HIV-1--LTR stimulation was also achieved when IL-1beta, IL-6, and TNF-beta were added to macrophages with cross-linked costimulatory molecules. Contact between activated lymphocytes and macrophages is necessary to down-regulate inhibitory C/EBPbeta, thereby derepressing the HIV-1 LTR. Lymphocyte-derived cytokines activate NF-kappaB, further enhancing the HIV-1 LTR.