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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2008 Aug 21;105(34):12509–12514. doi: 10.1073/pnas.0805504105

Erythromycin derivatives inhibit HIV-1 replication in macrophages through modulation of MAPK activity to induce small isoforms of C/EBPβ

Iwao Komuro *,†, Toshiaki Sunazuka ‡, Kiyoko S Akagawa *,‡, Yasuko Yokota *, Aikichi Iwamoto †, Satoshi Ōmura ‡,§
PMCID: PMC2527942  PMID: 18719105

Abstract

Macrophages (MΦs) are a major source of HIV-1 especially in patients with tuberculosis. There are MΦs that are permissive and those that restrict HIV-1. Regulation of hematopoietic cell kinase (Hck) activity and selective expression of CCAAT enhancer binding protein β (C/EBPβ) isoforms greatly contribute to determine distinct susceptibility of MΦs to HIV-1. Resistance is attributable to reduced expression of Hck and augmented expression of an inhibitory small isoform of C/EBPβ. Derivatives of erythromycin A (EMA) EM201 and EM703 inhibit the replication of HIV-1 in tissue MΦs, at posttranscriptional and translational levels. We demonstrate that EM201 and EM703 convert tissue MΦs from HIV-1 susceptible to HIV-1 resistant through down-regulation of Hck and induction of small isoforms of C/EBPβ. These drugs inhibit p38MAPK activation which is expressed only in susceptible tissue MΦs. Activated CD4+T cells stimulate the viral replication in HIV-1 resistant MΦs through down-regulation of small isoforms of C/EBPβ via activation of ERK1/2. EM201 and EM703 can inhibit the MAPK activation and inhibit the burst of viral replication produced when CD4+T cells and MΦs interact. These EM derivatives may be highly beneficial for repression of residual HIV-1 in the lymphoreticular system of HIV-1-infected patients and offer great promise for the creation of new anti-HIV drugs for the future treatment of AIDS patients.

Keywords: AIDS, macrolides, Hck

At least 65 million people have been infected with HIV and AIDS has killed 25 million people since 1981. By 2007, worldwide, 39.5 million individuals were living with HIV, with 4.3 million new infections and 2.9 million deaths occurring in 2006 (http://data.unaids.org/pub/EpiReport/2006/02-Global_Summary_2006_EpiUpdate_eng.pdf). In developed countries, anti-HIV-1 therapy—highly active antiretroviral therapy (HAART)— potently inhibits HIV-1 replication, reduces viral antigenemia, and prolongs the survival of patients (1, 2). In contrast, patients in developing countries generally cannot use HAART therapy because of its high cost and the sheer number of patients. Furthermore, HAART therapy cannot remove HIV-1-infected latent memory T cells and monocytes (Mos)/macrophages (MΦs) in some lymphoreticular tissue, residual cells having the potential to become a viral resource capable of spreading new viral particles (3, 4). Therefore, the development of new drugs to improve and extend HAART therapy, particularly in countries in the developing world, is greatly and urgently needed.

Mos/MΦs are a major target of HIV-1 infection and serve as a reservoir for viral persistence and a chronic source of infectious virus in vivo (5). Most tissue MΦs are permissive to M-tropic virus entry and release a small number of virus particles in the asymptomatic carrier but they occasionally produce a large number of viral particles in the AIDS patients or HIV-1 patients with pulmonary tuberculosis (TB) or those whose conditions are complicated with opportunistic infection (3). TB markedly increases HIV-1 replication and mutation in the lung and is associated with an acceleration of AIDS (6, 7). The alveolar MΦ is the major cell type in which HIV-1 replication occurs during TB (8, 9). Thus MΦ is a key factor in the control of HIV-1 suffering.

We and others have previously demonstrated that expression of tyrosine kinase hematopoietic cell kinase (Hck) and relative amounts of a large isoform (37-kDa) to a small isoform (23-kDa) (L/S ratio) of transcription factor CCAAT enhancer binding protein β (C/EBPβ) play critical roles in M-tropic HIV-1 production in tissue MΦs (8, 10–13). We have also reported that modulation of the expression of Hck and the L/S ratio of C/EBPβ by treatment with antisense oligonucleotides can convert the phenotype of HIV-1 suceptibility in MΦs (10). These studies suggest that, not only anti-HIV-1 drugs that directly affect the virus (such as RT inhibitor or protease inhibitors), but also drugs that can convert the phenotype of tissue MΦs from “susceptible” to “resistant” by down-regulating the expression of Hck and enhancing the expression of small isoforms of C/EBPβ may be useful to help control HIV-1 replication in AIDS patients.

Macrolides with a 14-membered ring structure, such as erythromycin A (EMA), clarithromycin (CAM), or roxithromycin (RXM), are well known antibacterial drugs. Recently, these antibiotics have been shown to be efficacious against incurable chronic inflammatory airway disease, such as diffuse panbronchiolitis (DPB) (14, 15). This therapeutic efficacy is thought to be caused by either anti-inflammatory or immunomodulatory activity of the macrolide antibiotics, which can act on many cells, including epithelial cells, neutrophiles, monocytes/MΦs, and T cells (16–23). On the basis of this knowledge, we chemically modified EMA to obtain derivatives with both stronger capability for promoting monocyte-to-MΦ differentiation and no antibacterial activity. Among the derivatives, 8,9-anhydroerythromycin A 6,9-hemiketal (EM201), obtained by mild acid treatment of EMA, already known as an internal metabolite of EMA, showed a strong promotional effect on MΦ differentiation and possessed weak antimicrobial activity (24). Furthermore, the 12-membered pseudoerythromycin A (EM703) was both remarkably active and free of any antibacterial activity (25) and was known to exhibit a prophylactic effect on lung injury in vivo against a bleomycin-induced acute lung injury in the rat model, similar to EMA (26).

In this study, we show that both EM201 and EM703 are good lead candidates for drugs that can inhibit M-tropic HIV-1 replication in tissue MΦs by a new way of converting their phenotype from HIV-1-susceptible to HIV-1-resistant, through down-regulation of Hck and the induction of small isoforms of C/EBPβ via modulation of the activation of MAPKs.

Results

Effects of EM Derivatives on Viral Replication and Multinucleated Giant Cell Formation in M-Tropic HIV-1-Infected M-MΦs.

We first examined whether EM derivatives (Fig. 1) have an ability to inhibit M-tropic HIV-1 replication in macrophage colony-stimulating factor (M-CSF)-induced monocyte-derived MΦs (M-MΦs), which express a high level of Hck and a large isoform of C/EBPβ, are susceptible to M-tropic HIV-1 replication, and whether they form multinucleated giant cells (MGC) by cell-to-cell fusion at 4–7 d after infection (10, 27). EM201 and EM703 (30 μM) completely inhibited viral replication and MGC formation at 7 d after infection, while EMA, CAM, and EM202 did not (Fig. 2A). PCR using a primer pair designed from the HIV-1 LTR region of HIV-1BaL DNA at 2 d after infection showed that the DNA replication at first replicon was observed at similar levels in all of the M-MΦs. At 7 d after infection, however, the levels of viral DNA in M-MΦs treated with EMA, EM202, or DMSO (solvent) alone increased, whereas those in M-MΦs treated with EM201and EM703 remained low, at levels similar to those observed at 2 d after infection (Fig. 2A).

Fig. 1.

Fig. 1.

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Structure of EM derivatives.

Fig. 2.

Fig. 2.

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Screening of EM derivatives that show an inhibitory effect on HIV-1BaL replication in M-MΦs. (A) Effects of 30 μM of EMA (EM), CAM, EM201 (201), EM202 (202), and EM703 (703) on viral replication and MGC formation (Magnification, ×100) at 7 d after infection. The data of viral production were shown as the percentage of p24 antigen in control (DMSO alone) M-MΦs. The levels of viral DNA were assayed at 2 and 7 d after infection. (B) Kinetics of viral production in HIV-1 infected M-MΦs treated with 30 μM of EM derivatives. (C) Dose-response effects of EM derivatives on HIV-1 replication in M-MΦs at 14 d after infection. (D) EM derivatives (30 μM) do not change the resistant phenotype against HIV-1 infection in GM-MΦs. The data shown are representative one of five independent experiments.

EM201 and EM703 (30 μM) persistently inhibited viral replication at 14 d (Fig. 2B), and inhibition was observed even at 21 d after infection (data not shown). EM201 and EM703 strongly inhibited HIV-1BaL replication, even at 3 μM, and p24 levels were ∼4% of those in cells treated with DMSO alone at 14 d after infection (Fig. 2C). EMA and EM202 induced inhibition of viral replication at higher concentration (>300 μM). However, the reduction curves in cells were similar to those in DMSO-treated cells (Fig. 2C), indicating the effects are mainly because of DMSO toxicity. In contrast, CAM partially but significantly inhibited HIV-1Bal replication at 10–30 μM at 10 and 14 d after infection (Figs. 2 B and C), and it is impossible to deny that CAM itself can inhibit HIV-1 replication.

EM201 and EM703 Modulate the Expression of Hck and C/EBPβ Proteins in HIV-1BaL Infected M-MΦs.

To examine the possibility that EM201 and EM703 inhibit HIV-1 replication in M-MΦs via modulation of the expression of Hck and C/EBPβ, expression of these proteins in HIV-1 infected M-MΦs treated with 30 μM EM derivatives was examined by immunoblots at 2 d after infection. The levels of Hck protein in M-MΦs treated with EM201 and EM703 strongly decreased to one-seventh and one-ninth of that in M-MΦs treated with DMSO alone, respectively (Fig. 3A). Conversely, the small isoform of C/EBPβ protein was strongly induced in M-MΦs treated with EM201 and EM703, the levels increasing to 25- to 40-fold of that in M-MΦs treated with DMSO alone, with the L/S ratio of C/EBPβ markedly decreasing from 12.6 to 0.3 and 0.5, respectively (Fig. 3A).

Fig. 3.

Fig. 3.

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Effects of EM derivatives on the expression of Hck and C/EBPβ in HIV-1BaL-infected M-MΦs and GM-MΦs. Immunoblots of Hck and C/EBPβ in M-MΦs (A) and GM-MΦs (B) at 2 d after infection. EMA (EM), EM201 (201), EM202 (202), EM703 (703), and CAM were added at 30 μM. The relative amounts of Hck and C/EBPβ were measured using National Institutes of Health image software (PSL; photo stimulating luminescence, A/mm2). The relative amounts of the large band to the small band (L/S ratio) of C/EBPβ were calculated using PSL values of 37 kDa and 23 kDa of C/EBPβ isoforms and are shown at the bottom of each figure. The data shown are representative of one of three independent experiments.

EMA and EM202 did not affect the expression of Hck and C/EBPβ and consequently did not inhibit viral replication (Fig. 3A). Similarly CAM, which did not show inhibitory activity during the early phase of infection, did not significantly affect expression of either Hck or C/EBPβ at 2 d after infection.

EM201 and EM703 Change Neither the Expression of Hck and C/EBPβ Proteins Nor the Resistant Phenotype Against HIV-1 Infection in GM-MΦs.

Granulocyte-macrophage CSF (GM-CSF)-induced monocyte-derived MΦ (GM-MΦ) is HIV-1 resistant and does not stimulate the replication of M-tropic HIV-1and MGC formation. This is because GM-MΦs express a high level of short isoforms of C/EBPβ and a low level of Hck, and HIV-1 infection drastically increases the expression of a short isoform of C/EBPβ but decreases that of Hck (10). We examined the effects of EM derivatives on viral replication and the expression of Hck and C/EBPβ in HIV-1BaL-infected GM-MΦs. Even at 14 d after infection, we found that GM-MΦs treated with various kinds of EM derivatives (including EM201 and EM703) did not stimulate viral replication (Fig. 2D) or MGC formation (data not shown). Consistent with the lack of change in HIV-1 resistant phenotype, all of the EM derivatives did not affect the expression of Hck and C/EBPβ protein in GM-MΦs (Fig. 3B).

p38MAPK Inhibitor, but Not ERK1/2 Inhibitor, Inhibits Viral Replication in M-Tropic HIV-1 Infected M-MΦs via Reduced Expression of Hck and Increased Expression of a Small Isoform of C/EBPβ.

Previous reports have shown that the replication of M-tropic HIV-1 in tissue MΦs requires the activation of p38MAPK (28) and that ERK1/2 mediates the activation of C/EBPβ (29, 30). We consequently examined the activation of MAPKs in HIV-1 susceptible M-MΦs and HIV-1 resistant GM-MΦs. Expressions of total and phosphorylated forms of p38MAPK in M-MΦs were higher than those in GM-MΦs before HIV-1BaL infection (Fig. 4A). After infection, the phosphorylated form was augmented in M-MΦs but not in GM-MΦs (Fig. 4A). In contrast to p38MAPK, the expressions of total and phosphorylated forms of ERK1/2 in M-MΦs were lower than those in GM-MΦs before infection, but the expression was unchanged in both MΦs after infection (Fig. 4A). Consistent with the augumented activation of p38MAPK in M-MΦs, addition of p38MAPK inhibitor SB203580 (at 10 μM) completely suppressed viral replication and MGC formation in HIV-1BaL-infected M-MΦs (Fig. 4B).

Fig. 4.

Fig. 4.

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Effects of p38 MAPK inhibitor and ERK1/2 inhibitor on viral replication and expression of Hck and C/EBPβ in HIV-1BaLinfected M-MΦs. (A) Immunoblot analysis of total and phosphorylated forms of p38MAPK and ERK1/2 in M-MΦs and GM-MΦs before and 2 d after infection. (B) Kinetic analysis of viral replication and morphology in HIV-1BaL-infected M-MΦs treated with 10 μM of SB203580 or PD98059. (C) Immunoblot analysis of Hck and C/EBPβ in HIV-1BaL-infected M-MΦs treated with various concentrations of SB203580 or PD98059 at 2 d after infection. (D) The relative amounts of Hck and L/S ratio of C/EBPβ in the cells or the phosphorylated protein P to the total protein T (P/T ratio) of p38 MAPK and ERK1/2 in immunoblot analysis shown in c calculated as described in Fig. 3. (E) Viral production in HIV-1BaL-infected M-MΦs treated with various concentrations of SB203580 or PD98059. The data shown are representative of one of three independent experiments.

We subsequently investigated whether the inhibitory activity of SB203580 on viral replication in HIV-1BaL-infected M-MΦs is mediated through modulation of the expression of Hck and C/EBPβ protein. SB203580 not only inhibited the phosphorylation of p38MAPK but also reduced the expression of Hck and increased the expression of the small isoform of C/EBPβ to mimic the inhibitory effect on viral replication (Fig. 4 C–E). Conversely, the ERK1/2 inhibitor PD98059 affected neither viral replication nor the expression of Hck and C/EBPβ protein (Fig. 4 B–E).

EM201 and EM703 Inhibit Viral Replication in M-Tropic HIV-1 Infected M-MΦs via Inhibition of p38MAPK Activation.

The above results suggest that EM201 and EM703 inhibit M-tropic HIV-1 replication via inhibition of p38MAPK activation. To help confirm this hypothesis, we examined the effect of EM201 or EM703 (30 μM) on the phosphorylation of p38MAPK and ERK1/2 in M-MΦs by immunoblot. EM201 and EM703, but not EMA, reduced phosphorylation of p38MAPK but enhanced the phosphorylation of ERK1/2 in HIV-1-infected M-MΦs at 2 d after treatment (Fig. 5).

Fig. 5.

Fig. 5.

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Effects of EM derivatives on the phosphorylation of p38 MAPK and ERK1/2 in M-MΦs and GM-MΦs. (A) Immunoblot analysis of Hck and C/EBPβ, and the phosphorylation of p38 MAPK and ERK1/2 in M-MΦs and GM-MΦs treated with 30 μM of EMA (EM), EM201 (201), EM703 (703), 10 μM of SB203580 (SB), or PD98059 (PD), and DMSO alone at 2 d after HIV-1 infection. (B) The relative amounts of Hck and L/S ratio of C/EBPβ or P/T ratio of p38 MAPK and ERK1/2 in immunoblot analysis shown in a are calculated as described in Fig. 3. The data shown are representative of one of three independent experiments.

As described above, EM201 and EM703 did not affect the HIV-1-resistant phenotype of GM-MΦs (Figs. 2D and 3B). Consistent with the results, none of the EM derivatives significantly affected the phosphorylation pattern of p38MAPK and ERK1/2 in HIV-1-infected GM-MΦs (Fig. 5).

Activated CD4+T Cells Stimulate Viral Replication in M-Tropic HIV-1 Infected GM-MΦs via Down-Regulation of a Small Isoform of C/EBPβ.

Recently, Hosino et al. (9) reported a phenotypical change of human alveolar MΦs (A-MΦs) from resistant to susceptible for HIV-1 replication caused by the addition of activated lymphocytes. The change was brought about by decreased expression of a small isoform of C/EBPβ (9). In line with this report, the addition of activated CD4+T cells to HIV-1BaL-infected GM-MΦs stimulated marked viral replication (Fig. 6A), with MGC formation and clusters of GM-MΦs with CD4+ T cells (data not shown) at 10–14 d after infection. The amounts of viral DNA in the GM-MΦs increased at 2–7 d after infection (Fig. 6B). In GM-MΦs stimulated with activated CD4+ T cells, expression of the small isoform of C/EBPβ protein significantly decreased whereas the L/S ratio of C/EBPβ increased (from 0.57 to 3.6) at 2 d after infection (Fig. 6 C and D). Expression of Hck in the GM-MΦs, however, did not change significantly, even after stimulation with activated T cells and was very low compared with that in M-MΦs (Fig. 6 C and D).

Fig. 6.

Fig. 6.

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Augmentation of M-tropic HIV-1 production in GM-MΦs stimulated with CD4+ T cells, and the suppressive effects of EM201 and EM703 on viral replication through the induction of small isoforms of C/EBPβ via inhibition of phosphorylation of ERK1/2. M-MΦs and GM-MΦs were infected with HIV-1BaL. Part of HIV-1-infected GM-MΦs were stimulated with activated CD4+ T cells and incubated with or without 30 μM of EMA (EM), EM201 (201), or EM703 (703), 10 μM of SB203580 (SB), or PD98059 (PD), and DMSO alone. (A) Kinetic analysis of HIV-1 replication. (B) Levels of viral DNA at 2 and 7 d after infection. (C) Immunoblot analysis of Hck, C/EBPβ, and phosphorylation of p38 MAPK and ERK1/2 at 2 d after infection. (D) The relative amounts of Hck and L/S ratio of C/EBPβ or P/T ratio of p38 MAPK and ERK1/2 in immunoblot analysis shown in c calculated as described in Fig. 3. (M) M-MΦ; GM, GM-MΦ; T, T cells; 201, EM201; 703, EM703; SB, SB203580; and PD, PD98059. The data shown are representative of one of three independent experiments.

Activated CD4+T Cells Down-Regulate the Small Isoform of C/EBPβ in M-Tropic HIV-1-Infected GM-MΦs via Augmentation of ERK1/2 Phosphorylation.

As described above, activation of p38MAPK but not ERK1/2 is critical for HIV-1 replication in M-MΦs. However, the p38MAPK inhibitor, SB203580, did not inhibit viral replication in GM-MΦs stimulated with activated CD4+T cells (Fig. 6A). Instead, the ERK1/2 inhibitor PD98059 completely inhibited viral replication (Fig. 6A) and suppressed the level of viral DNA to that observed in the culture of GM-MΦs alone in which viral replication was absent (Fig. 6B). Upon examination of the phosphorylation of p38MAPK and ERK1/2 in HIV-1BaL-infected GM-MΦs stimulated with activated CD4+ T cells, the phosphorylation ratio of ERK1/2 but not of p38MAPK significantly increased in GM-MΦs stimulated with activated CD4+ T cells, compared with that in GM-MΦs alone. Addition of PD98059 not only inhibited the phosphorylation of ERK1/2 but also increased expression of the small isoform of C/EBPβ, while markedly decreasing the L/S ratio of C/EBPβ from 3.6 to 0.82 (Fig. 6 C and D). The addition of SB203580 did not affect the expression of C/EBPβ. The expression of Hck was unaffected by treatment with either of the two inhibitors (Fig. 6 C and D).

EM201 and EM703 Inhibit M-Tropic HIV-1 Replication in GM-MΦs Stimulated with Activated CD4+ T Cells via Inhibition of the Activation of ERK1/2 and Augmentation of the Expression of the Small Isoform of C/EBPβ.

In examining whether EM201 and EM703 can inhibit viral replication in M-tropic HIV-1-infected GM-MΦs stimulated with activated CD4+T cells, addition of EM201 and EM703 (30 μM) completely inhibited viral replication (Fig. 6A) and MGC formation (data not shown). The levels of HIV-1 DNA observed were very low, the same as those seen in the culture of GM-MΦs alone (Fig. 6B).

We subsequently examined the effects of EM201 and EM703 on the expression of Hck and C/EBPβ and on the phosphorylation of p38MAPK and ERK1/2 in HIV-1BaL-infected GM-MΦs stimulated with activated CD4+ T cells at 2 d after infection by immunoblot. Treatment with EM201 and EM703 did not change the levels of Hck protein, but increased levels of the small isoform of C/EBPβ protein and the L/S ratio of C/EBPβ decreased from 3.6 to 0.45 (EM201) and 0.44 (EM703) (Fig. 6 C and D). The phosphorylation level of ERK1/2 decreased following treatment with EM201 and EM703, but that of p38MAPK remained unchanged (Fig. 6 C and E).

Discussion

In this study, we demonstrated that two EMA derivatives, EM201and EM703, can inhibit the replication of M-tropic HIV-1 in tissue MΦs at the posttranscriptional and translational levels, but do not affect viral entry and first DNA replicon. The inhibition is caused by a new means of converting the phenotype of tissue MΦs, from HIV-1 susceptible to HIV-1 resistant, via down-regulation of Hck and induction of the small isoform of C/EBPβ through modulation of the activation of MAPKs. Consistent with the previous report (9), we showed that HIV-1-resistant GM-MΦs, require stimulation with activated CD4+T cells to produce vigorous virus production. This is mediated by down-regulation of the expression of the small isoform of C/EBPβ. Both EM201 and EM703 potently inhibit viral replication, not only in M-MΦs but also in GM-MΦs stimulated with activated CD4+T cells via inhibiting the down-regulation of the expression of the small isoform of C/EBPβ.

Both EM201 and EM703 change the phenotype only of HIV-1-susceptible MΦs. They do not affect the phenotype of HIV-1-resistant GM-MΦs. HIV-1 susceptibility and the expression of Hck and C/EBPβ proteins in A-MΦs from normal healthy volunteers are the same as those in GM-MΦs (8, 10). Therefore EM201 and EM703 do not change the resistant phenotype of A-MΦs. This would be beneficial for healthy A-MΦs in the HIV-1 carrier, by maintaining resistance against HIV-1 replication.

In the present study, we demonstrated that activation of p38MAPK and ERK1/2 play a critical role in HIV-1 production via down-regulation of the small isoform of C/EBPβ in HIV-1-infected-M-MΦs and -GM-MΦs stimulated with activated CD4+T cells, respectively. This study shows that different MAPKs play crucial roles in HIV-1 production in different types of tissue MΦs. P38MAPK activation in HIV-1-infected M-MΦs link to the augmented expression of Hck and the maintenance of the low level of the small isoform of C/EBPβ. We previously reported that reduced expression of Hck in M-MΦs with antisense oligonucleotide for Hck stimulates the induction of the short isoform of C/EBPβ and inhibits the viral replication (10). Our present results, taken together with the previous study, show the unique evidence that the p38MAPK signal cascade is upstream of Hck expression and is linked to down-regulation of the small isoform of C/EBPβ in HIV-1-susceptible M-MΦs. However, ERK1/2-mediated down-regulation of the small isoform of C/EBPβ in HIV-1-infected GM-MΦs stimulated with activated CD4+T cells does not link to Hck expression.

Interestingly, EM201 and EM703, in contrast to existing MAPK inhibitors, can inhibit viral replication via prevention of the activation of respective MAPKs in both HIV-1-infected-M-MΦs and -GM-MΦs stimulated with activated T cells, where different MAPKs play a critical role for viral replication. Such a novel and unique suppressive mechanism of EM201 and EM703 on HIV-1 replication in tissue MΦs may be useful for the future treatment of AIDS patient.

The anti-HIV-1 activity of EM201 and EM703 does not relate to their antibiotic activity, because they have only weak (EM201) or completely lack (EM703) such antibiotic activity (24, 25). At present, we do not know what kind of structure activity relationships exist in EM201 and EM703. Recently, calmodulin- and calmodulin-dependent protein kinase-II (CaMK-II)-dependent activation of p38MAPK has been reported in HIV accessory protein, Tat-induced IL-10 expression in normal human monocytes (31). EM201 and EM703 are known to act as inhibitors of intracellular Ca2+ level and Ca2+ oscillation (21, 32, 33). These characteristics may contribute to the novel anti-HIV-1 mechanism of EM201 and EM703.

Macrolides, such as EMA and CAM, are known to be specifically accumulated into tissue MΦs and stay stable at high levels for long periods because of a low rate of breakdown and excretion (34). EM201 and EM703 potently inhibit HIV-1 replication in MΦs at low levels, such as 30 μM, that correspond to the concentration of EMA or CAM in MΦs after oral intake of EMA, 400 mg or CAM 200 mg/day (usual doses are 1600 mg and 400 mg/day, respectively). Furthermore, inhibition of viral replication can be observed at lower concentrations, such as 3 μM, which is sustained for 2–14 days after infection. These findings offer advantages with respect to drug specificity and reduction of drug toxicity. In addition, these new macrolides are derived from EMA (24, 25) and would be very inexpensive. Thus, these substances offer great potential for the creation of new anti-HIV-1 drugs for the future treatment of AIDS patients.

Materials and Methods

Erythromycin Derivatives.

EMA was purchased from Sigma-Aldrich. CAM was supplied by Taisho Pharmaceutical. EM201, EM202, and EM703 were prepared as described previously (24, 25).

Preparation and Culture of MΦs.

Monocytes (Mos) and Mo-derived Mp were prepared as described previously (10, 35). M-CSF-induced monocyte-derived MΦs and GM-CSF-induced monocyte-derived MΦs were called M-MΦs and GM-MΦs, respectively. [see supporting information (SI) Materials and Methods for a detailed description].

HIV-1 Strain and Infection.

M-tropic HIV-1 strain, HIV-1BaL, was collected from culture supernatant of the HIV-1 strain-infected M-MΦs as a viral resource. Mo-derived MΦs were incubated for 2 h at 37°C with 100 pg/ml p24 antigen of DNase-treated viral supernatant (p24, the 50% tissue culture infective dose (TCID50) and multiplicity of infection (MOI) are 50 ng/ml, ∼3,000 and 0.05, respectively) and then cultured in RPMI MEDIUM 1640 containing 10% FCS and CSF. If necessary, the viral inoculum was pretreated with 100 μM AZT for 2 h at 4°C (5). Fresh culture medium containing CSF was added every 3–4 d (20% of the volume). Heat-inactivated virus (1 h, 56°C) was used as negative control. Viral production was assayed by sequential measurement of p24 antigen in supernatants by an ELISA using a combination of two antibodies; anti-gag-p24 monoclonal antibody (Nu24) and peroxidase-labeled 10B5 (36), or the RETRO-TEK HIV-1 p24 antigen ELISA kit for high-affinity detection of low levels of p24 antigen (ZeptoMetrix, Buffalo, New York).

Coculture of HIV-1 Infected GM-MΦs with the Activated CD4+T Cells.

CD4+ T cells were positively isolated from CD14− PBMCs using a MACS with anti-CD4 mAb coated microbeads. The selected population was >93% positive for CD3 and CD4. Activated CD4+ T cells were prepared by stimulation with PHA and cultured with IL-2 (30 unit/ml) (Genzyme). GM-MΦs were incubated for 2 h at 37°C with 100 pg/ml p24 antigen of DNase-treated viral supernatant, washed twice, and then cocultured with the activated CD4+ T cells in the presence of IL-2.

Detection of HIV-1 DNA by Nested PCR.

Detection of HIV-1 DNA by nested PCR was performed as described previoiusly (10). HIV LTR and gag primers were JAM 62 and JAM 65. For the nested PCR, JAM 63 and JAM 64 were used as internal primers (36). (see SI Materials and Methods for a detailed description).

Immunblot Analysis.

Immunoblot analysis was performed as described in ref. 10. Antibodies against the following proteins were used: rabbit polyclonal antibody against Hck (N-30), C/EBPβ (C-19) (Santa Cruz Biotechnology), phospho-specific (Tyr 182) p38 mitogen-activated protein kinase (P38MAPK) (no. 9211), p38 MAPK (no. 9212), phospho-specific (Tyr 204) phosphorylated extracellular signal-regulated kinases (ERK)1/2 (no. 9101), ERK1/2 antibody (no. 9102) (New England BioLabs), or normal rabbit IgG. Horseradish peroxidase-conjugated goat anti-rabbit IfG (sx-2030)(Santa Cruz Biotechnology) was used as secondary Ab. The blots were visualized with Amersham ECL Reagent on Hyper ECL-film (Amersham). (see SI Materials and Methods for a detailed description).

Statistical Analysis.

Statistical analysis of the data were performed using Student's t test. P-values <0.01 were considered significant. The experiments shown are representatives of three to seven independent experiments.

Supplementary Material

Supporting Information
supp_105_34_12509__index.html (681B, html)

Acknowledgments.

This work was supported in part by grants for Research on Health Sciences Focusing on Drug Innovation from the Japan Health Sciences Foundation and the Ministry of Health, Labor and Welfare of Japan.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0805504105/DCSupplemental.

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