Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression.

Stress, either physical or psychological, can have a dramatic impact on the immune system. Little progress, however, has been made in understanding stress-induced immune suppression. We report here that mice subjected to chronic 12-hour daily physical restraint for two days significantly increased the expression of Toll-like receptor 4 (TLR4). Interestingly, TLR4-deficient mice are resistant to stress-induced lymphocyte reduction. In addition, restraint stress caused dramatic decrease in T help 1 (Th1) cytokine IFN-gamma and IL-2 levels but increase in Th2 cytokine IL-4 in wild type mice. Moreover, the restraint stress significantly inhibits changes of Th1 and Th2 cytokines in TLR4-deficient mice compared with the wild type mice. Therefore, stress modulates the immune system through a TLR4-dependent mechanism.

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.

IFN-alpha beta secreted during infection is necessary but not sufficient for negative feedback regulation of IFN-alpha beta signaling by Mycobacterium tuberculosis.

IFN-alphabeta functions in the transition from innate to adaptive immunity and may impinge on the interaction of Mycobacterium tuberculosis with its host. Infection by M. tuberculosis causes IFN-alphabeta secretion and down-regulation of IFN-alphabeta signaling in human APC and the human monocytic cell line THP-1, which provides a model for these studies. Neutralization of secreted IFN-alphabeta prevents inhibition of IFN-alpha signaling during infection, but several lines of evidence distinguish inhibition due to infection from a negative feedback response to only IFN-alphabeta. First, greater inhibition of IFN-alpha-stimulated STAT-1 tyrosine phosphorylation occurs 3 days postinfection than 1 or 3 days after IFN-alphabeta pretreatment. Second, LPS also induces IFN-alphabeta secretion and causes IFN-alphabeta-dependent down-regulation of IFN-alpha signaling, yet the inhibition differs from that caused by infection. Third, IFN-alpha signaling is inhibited when cells are grown in conditioned medium collected from infected cells 1 day postinfection, but not if it is collected 3 days postinfection. Because IFN-alphabeta is stable, the results with conditioned medium suggest the involvement of an additional, labile substance during infection. Further characterizing signaling for effects of infection, we found that cell surface IFN-alphabeta receptor is not reduced by infection, but that infection increases association of protein tyrosine phosphatase 1c with the receptor and with tyrosine kinase 2. Concomitantly, IFN-alpha stimulation of tyrosine kinase 2 tyrosine phosphorylation and kinase activity decreases in infected cells. Moreover, infection reduces the abundance of JAK-1 and tyrosine-phosphorylated JAK-1. Thus, the distinctive down-regulation of IFN-alpha signaling by M. tuberculosis occurs together with a previously undescribed combination of inhibitory intracellular events.

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.

Aerosolized gamma interferon (IFN-gamma) induces expression of the genes encoding the IFN-gamma-inducible 10-kilodalton protein but not inducible nitric oxide synthase in the lung during tuberculosis.

Gamma interferon (IFN-gamma) is critical in the immune response against Mycobacterium tuberculosis. In an ongoing trial of aerosol IFN-gamma in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-gamma treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro, M. tuberculosis induced IP-10, and IFN-gamma stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-gamma. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-gamma did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-gamma. However, only 15 genes were differentially regulated by IFN-gamma. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-gamma treatment increases expression of few genes in the human lung.

Posttranscriptional inhibition of gene expression by Mycobacterium tuberculosis offsets transcriptional synergism with IFN-gamma and posttranscriptional up-regulation by IFN-gamma.

Host defense against Mycobacterium tuberculosis requires the cytokine IFN-gamma and IFN regulatory factor 1 (IRF-1), a transcription factor that is induced to high levels by IFN-gamma. Therefore, we chose to study regulation of IRF-1 expression as a model for effects of M. tuberculosis on response to IFN-gamma. We found that IRF-1 mRNA abundance increased far more than transcription rate in human monocytic THP-1 cells stimulated by IFN-gamma, but less than transcription rate in cells infected by M. tuberculosis. IFN-gamma stimulation of infected cells caused a synergistic increase in IRF-1 transcription, yet IRF-1 mRNA abundance was similar in uninfected and infected cells stimulated by IFN-gamma, as was the IRF-1 protein level. Comparable infection by Mycobacterium bovis bacillus Calmette-Guérin failed to induce IRF-1 expression and had no effect on the response to IFN-gamma. We also examined the kinetics of transcription, the mRNA t(1/2), and the distribution of IRF-1 transcripts among total nuclear RNA, poly(A) nuclear RNA, and poly(A) cytoplasmic RNA pools in cells that were infected by M. tuberculosis and/or stimulated by IFN-gamma. Our data suggest that infection by M. tuberculosis inhibits RNA export from the nucleus. Moreover, the results indicate that regulated entry of nascent transcripts into the pool of total nuclear RNA affects IRF-1 expression and that this process is stimulated by IFN-gamma and inhibited by M. tuberculosis. The ability of infection by M. tuberculosis to limit the increase in IRF-1 mRNA expression that typically follows transcriptional synergism may contribute to the pathogenicity of M. tuberculosis.

Inhibition of response to alpha interferon by Mycobacterium tuberculosis.

We previously reported that infection by Mycobacterium tuberculosis, the causative agent of tuberculosis, leads to secretion of alpha/beta interferon (IFN-alpha/beta). While IFN-alpha/beta ordinarily stimulates formation of signal transducer and stimulator of transcription-1 (STAT-1) homodimers and IFN-stimulated gene factor-3 (ISGF-3), only ISGF-3 is found in infected human monocytes and macrophages. We have now investigated the basis for this unusual profile of transcription factor activation and its consequences for regulation of transcription, as well as the impact of infection on response to IFN-alpha. After infection, IFN-alpha stimulation of STAT-1 homodimers is inhibited in monocytes and macrophages, while stimulation of ISGF-3 increases in monocytes but tends to decline in macrophages. Effects of infection on the abundance of ISGF-3 subunits, STAT-1, STAT-2, and interferon regulatory factor 9, and on tyrosine phosphorylation of STAT-1 and STAT-2 explain the observed changes in DNA-binding activity, which correlate with increased or inhibited transcription of genes regulated by ISGF-3 and STAT-1. Infection by Mycobacterium bovis BCG does not inhibit IFN-alpha-stimulated tyrosine phosphorylation of STAT-1, formation of homodimers, or transcription of genes regulated by STAT-1 homodimers, suggesting that inhibition of the response to IFN-alpha/beta by M. tuberculosis is an aspect of pathogenicity. Thus, this well-known feature of infection by pathogenic viruses may also be a strategy employed by pathogenic bacteria.

Host defense responses to infection by Mycobacterium tuberculosis. Induction of IRF-1 and a serine protease inhibitor.

Alveolar macrophages and newly recruited monocytes are targets of infection by Mycobacterium tuberculosis. Therefore, we examined the expression of interferon regulatory factor 1 (IRF-1), which plays an important role in host defense against M. tuberculosis, in undifferentiated and differentiated cells. Infection induced IRF-1 in both. IRF-1 from undifferentiated, uninfected monocytic cell lines was modified during extraction to produce specific species that were apparently smaller than intact IRF-1. After infection by M. tuberculosis or differentiation, intact IRF-1 was recovered. Subcellular fractions were assayed for the ability to modify IRF-1 or inhibit its modification. A serine protease on the cytoplasmic surface of an organelle or vesicle in the "lysosomal/mitochondrial" fraction from undifferentiated cells was responsible for the modification of IRF-1. Thus, the simplest explanation of the modification is cleavage of IRF-1 by the serine protease. Recovery of intact IRF-1 correlated with induction of a serine protease inhibitor that was able to significantly reduce the modification of IRF-1. The inhibitor was present in the cytoplasm of M. tuberculosis-infected or -differentiated cells. It is likely that induction of both IRF-1 and the serine protease inhibitor in response to infection by M. tuberculosis represent host defense mechanisms.