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Originally published online as doi:10.1189/jlb.0306206 on August 30, 2006

Published online before print August 30, 2006
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(Journal of Leukocyte Biology. 2006;80:1136-1144.)
© 2006 by Society for Leukocyte Biology

Mycobacterium avium-induced SOCS contributes to resistance to IFN-{gamma}-mediated mycobactericidal activity in human macrophages

Nancy Vázquez*,1, Teresa Greenwell-Wild*, Sofia Rekka*, Jan M. Orenstein{dagger} and Sharon M. Wahl*

* Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland, USA; and
{dagger} Department of Pathology, George Washington University, Washington, DC, USA

1 Correspondence: Building 30, 30 Convent Dr., MSC 4352, OIIB NIDCR, NIH, Bethesda, MD 20892-4352. E-mail: nvazquez{at}mail.nih.gov

ABSTRACT

Mycobacterium avium is an opportunistic pathogen that commonly infects individuals colonized with HIV-1, although it is less frequent in the post-HAART era. These microorganisms invade macrophages after interacting with TLR2 and/or CD14 co-receptors, but signaling pathways promoting survival in macrophages are not well defined. Although IFN-{gamma} plays an important role in protective immunity against bacterial infections, IFN-{gamma} responses are compromised in AIDS patients and evidence suggests that exogenous IFN-{gamma} is inadequate to clear the mycobacteria. To determine the mechanism by which M. avium survives intracellularly, even in the presence of IFN-{gamma}, we studied the effect of mycobacteria infection in macrophages during early IFN-{gamma} signaling events. M. avium infected cells exhibited a reduced response to IFN-{gamma}, with suppressed phosphorylation of STAT-1 compared with uninfected cells. Interaction of M. avium with macrophage receptors increased gene expression of the suppressors of cytokine signaling (SOCS) to diminish IFN responsiveness. Specifically, we observed an increase in mRNA for both SOCS-3 and SOCS-1, which correlates with elevated levels of SOCS protein and positive immunostaining in M. avium/HIV-1 co-infected tissues. We also linked the p38 MAPK signaling pathway to mycobacterial-induced SOCS gene transcription. The induction of SOCS may be part of the strategy that allows the invader to render the macrophages unresponsive to IFN-{gamma}, which otherwise promotes clearance of the infection. Our data provide new insights into the manipulation of the host response by this opportunistic pathogen and the potential for modulating SOCS to influence the outcome of M. avium infection in immunocompromised hosts.

Key Words: AIDS • STAT • HIV

INTRODUCTION

M. avium is a ubiquitous environmental microorganism [1 ], and infections with M. avium are not a cause for morbidity and mortality in the immunocompetent host. However, disseminated infections with this gram-positive facultative intracellular microorganism are usually a complication during HIV-1 infection [2 , 3 ]. Coinfection with opportunistic pathogens such as Mycobacterium avium complex (MAC) typically occurs in HIV-1 infected patients with a T cell count less than 100 CD4+ cells/mm3 and high viral burden [3 , 4 ]. Despite a transient immune reconstitution disease caused by M. avium complex presenting shortly after the introduction of HAART [5 ], infections with these bacteria become less frequent in long-term HAART-treated patients [6 ]. However, in regions of the world with limited access to HAART, MAC still represents a clinical burden. Analysis of tissue specimens revealed a marked increase in HIV-1 within macrophages, which are concordantly infected with one or more opportunistic microorganisms, when compared with tissues infected with HIV-1 alone, which suggests that not only does HIV-1 immunodeficiency increase susceptibility to opportunistic infections (OI) but that the opportunistic pathogens promote HIV-1 in a reciprocal relationship [7 ]. Therefore, co-infection must provide key signals that promote HIV infection/replication and M. avium survival. Although the vast majority of MAC infections are found in AIDS patients, individuals with predisposing pulmonary conditions, post-transplant immunosuppressed and elderly individuals are also susceptible to mycobacteria [8 ]. Studies by other investigators have shown that mycobacteria inactivate macrophage function and promote their own survival by preventing acidification/maturation of the phagosomes [9 ] and inhibiting IFN-{gamma} induced HLA-DR [10 ]. Mycobacteria also block the expression of co-stimulatory and adhesion molecules in monocytes as well as dampen cytokines that play an important role in host defense against Mycobacterium avium [11 , 12 ]. However, it is not completely clear how mycobacteria-induced signaling contributes to macrophage dysfunction. Individuals with IFN-{gamma} mediated immune inherited disorders and STAT-1 deficiencies are vulnerable to mycobacterial infections [13 14 15 16 ], emphasizing the important immunoregulatory role of IFN-{gamma} in anti-mycobacterial immune responses.

Although IFN-{gamma} confers protective immunity against mycobacterial infections [13 , 14 ], macrophages infected with mycobacteria can become refractory to this cytokine [10 , 17 , 18 ]. Stimulation of the IFN-{gamma} receptor by its cognate ligand leads to receptor oligomerization, followed by activation and phosphorylation of the non-receptor tyrosine Janus kinases JAK-1 and JAK-2. Activated JAKs then phosphorylate the intracellular domain of the interferon-gamma receptor 1 (IFN-{gamma}-R1) chain, generating a temporary docking site for STAT-1. In turn, binding of STAT-1 to the IFN-{gamma} receptor leads to STAT-1 phosphorylation by JAKs, inducing STAT-1 dimerization through Src-homology (SH2) domains. This is followed by nuclear translocation of STAT-1 homodimers, which regulate IFN-{gamma} induced gene transcription by binding to gamma-activated sequences (GAS) [19 ]. As a counterbalance, the suppressor of cytokine signaling (SOCS) family, also known as cytokine-inducible SH2 domain-containing protein (CIS), JAK binding protein (JAB), or STAT-induced STAT inhibitors (SSI), function as negative regulators of cytokine and toll receptor (TLR)-induced signaling [20 , 21 ]. Importantly, the SOCS proteins have been shown to down-regulate the IFN-{gamma} signaling pathway [22 23 24 25 ].

To further elucidate how mycobacterial infections modulate human macrophage activity for its benefit, cells were infected with M. avium and analyzed for signal transduction and gene expression. Downstream events showed induction of mRNA for the inhibitors of IFN-{gamma}, SOCS-3, and SOCS-1. This rapid induction of SOCS was blocked by interfering with p38 MAPK signaling pathway preventing M. avium-induced SOCS gene expression and protein production. These results provide a potential mechanism by which M. avium suppresses host responses to interferons to generate the conditions that favor survival of the microorganism.

MATERIALS AND METHODS

Purification of human monocytes by counterflow centrifugal elutriation
Human peripheral blood mononuclear cells were obtained by leukapheresis from normal volunteers in the Department of Transfusion Medicine at the National Institutes of Health (Bethesda, MD) and diluted in endotoxin-free PBS without Ca2+ and Mg2+ (BioWhittaker, Walkersville, MD) for density sedimentation. The monocytes in the mononuclear cell layer were purified by counterflow centrifugal elutriation within 4 h after leukapheresis [26 ]. Freshly elutriated monocytes were resuspended in DMEM containing 2 mM L-glutamine and 50 µg/ml gentamicin (BioWhittaker), plated in 24- or 6-well plates (Corning Costar, Corning, NY) at 1.5 x 106 and 6 x 106 cells per well, respectively, and allowed to adhere for 2–4 h, and 10% fetal bovine serum (FBS) was added. The cells were allowed to differentiate into monocyte-derived macrophages (MDM) by culturing 6–7 days at 37°C with 5% CO2.

M. avium infection of monocyte-derived macrophages
M. avium, a virulent smooth, transparent morphotype strain 2-151, was grown, as described previously [27 , 28 ], and viable organisms were added to adherent MDM. Mycobacterial antigen (MAg) and lipoarabinomannan (LAM), both derived from M. avium, were generated as described previously [27 , 29 ]. MDM 7-day culture supernatants were removed and replaced with fresh complete medium (DMEM containing gentamicin, L-glutamine, and 10% FBS). M. avium was added at a ratio of 5:1 for 2 h, and excess bacteria was removed by collecting the 2 h supernatant and washing the adherent macrophages twice with medium. Cultures were treated with 25 µg/ml MAg or 8 µg/ml LAM [30 ]. In some experiments, cultures were pretreated with TNF-{alpha} (10 ng/ml) or IFN-{gamma} (10 ng/ml) (National Cancer Institute-Frederick Cancer Research and Development Center). Cells were fixed in 2.5% gluteraldehyde after cytokine/bacteria treatment and embedded in plastic; after which 1 µm, semi-thin sections were cut, stained with toluidine blue, and examined by light microscopy. Some cultures were treated with the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 (Calbiochem, San Diego, CA) for 60 min before adding M. avium.

Immunoprecipitation and Western blot analysis
Control and mycobacteria-infected macrophages were washed twice with PBS containing 0.1% Na3VO4. Whole cell lysates were generated by using a lysis buffer that consisted of 1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 10 mM NaF, 10 mM NaPPi, 2.5 nM EDTA, 1 mM Na3VO4, 0.2 mM 3, 4 dicloroisocoumarin, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml chymostatin, and 1x complete protease inhibitor (Boehringer Mannheim, Indianapolis, IN). Cell lysates were centrifuged at 12,000 rpm for 15 min. SOCS-3 was immunoprecipitated from cell lysates using an anti-SOCS-3 antibody (Fusion Antibodies, Belfast, Northern Ireland) and incubated with constant rotation at 4°C for 2 h. Immunoprecipitates were washed and then resuspended in reducing Laemmli buffer and analyzed using a 4–20% Tricine gel (Invitrogen, Carlsbad, CA), transferred to a nitrocellulose membrane and immunoblotted with anti-SOCS-3 biotin-conjugated antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). Membranes were incubated with streptavidin-HRP conjugate (Upstate Biotechnology, Lake Placid, NY) for 30 min and washed, and immunoblots were developed using Super-Signal substrate according to manufacturer’s instructions (Pierce, Rockford, IL).

Real-time PCR
Total cellular RNA was extracted from adherent control or infected macrophages with the RNeasy minikit (Qiagen, Valencia, CA). All samples were treated with DNase according to the manufacturer’s instructions (Qiagen). For RT-PCR, 1 µg of total RNA was used for reverse transcription by oligodeoxythymilic acid primer and the resulting cDNA amplified by PCR using the ABI 7500 Sequence Detector (Applied Biosystems, Foster City, CA). Amplification was performed using the Taqman expression assays for SOCS-1 (Hs00705164_s1), SOCS-3 (Hs00269575_s1), and GAPDH (Hs99999905_m1) as normalization control according to the parameters established by the manufacturer (Applied Biosystems). The data were examined using the 2-{Delta}{Delta}CT method [31 ], and results expressed as fold increase.

RNase protection assay (RPA)
Total cellular RNA was extracted from adherent control or infected macrophages as indicated above. For the RPA, 3 µg of RNA was evaluated using the Riboquant Multi-Probe RPA system h-SOCS-559927 probe set (BD PharMingen, San Diego, CA), and densities were normalized to the GAPDH gene using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Immunohistochemistry
Lymph node (LN) tissue biopsies obtained from patients with AIDS-defining opportunistic infection (OI), HIV-1-seropositive subjects without evidence of OI, and HIV-1 seronegative donors were fixed in 10% neutral buffered formalin, paraffin-embedded, and sectioned. Tissue sections were dewaxed/rehydrated and processed for antigen retrieval in a decloaking chamber (Biocare Medical, Concord, CA) in antigen unmasking solution (Vector Laboratories, Burlingame, CA) followed by cooling at room temperature. Endogenous peroxidase activity was blocked with 3% H2O2 in 50% methanol for 15 min. The sections were incubated with normal blocking serum (Vectastain Elite ABC Kit; Vector Laboratories) for 20 min, followed by incubation with anti-SOCS-3 antibody (5 µg/ml) (Santa Cruz Biotechnology) overnight at 4°C. The sections were incubated for 30 min with biotinylated secondary antibody, and immunoreactive staining was developed using streptavidin-peroxidase (Vector Laboratories) followed by diaminobenzidene (Liquid DAB substrate Kit, Zymed Laboratories, San Francisco, CA). Sections were counter-stained with Mayer’s hematoxylin. Immunohistochemical staining was also performed in the presence of isotype-matched control primary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) as control. Some tissue sections were processed for in situ hybridization for HIV RNA and M. avium detection as described previously [27 ].

Statistical analysis
Data are presented as mean ± SEM and analyzed for significance using Student t test.

RESULTS

IFN-{gamma} response in macrophages infected with M. avium
We have shown that in vitro mycobacterium infection of macrophages in the absence of T cells triggers an early response consistent with immune activation leading to transcriptional activation of multiple genes involved in regulation of inflammatory and immune responses [30 ]. However, in spite of this pro-active response [30 ], macrophage microbicidal activity is inefficient and mycobacteria thrive within their macrophage hosts. In vivo, CD4+ T cells and IFN-{gamma} are crucial to host anti-microbial responses, yet M. avium-infected macrophages in vitro appear refractory to treatment with IFN-{gamma} and as a consequence they are susceptible to infection and mycobacterial replication (Fig. 1 ). Cells were treated with medium alone, TNF-{alpha}, a cytokine with anti-mycobacterial capacity [18 ], or IFN-{gamma} for 18 h, infected with mycobacteria for 2 h, and nonadherent or noninternalized mycobacteria removed. Cultures were examined at different intervals post-infection for the presence of M. avium. Macrophages infected with M. avium show increased numbers of intracellular bacteria 10 days after infection, and also cell necrosis (Fig. 1B) , when compared with uninfected cells (Fig. 1A) . By comparison, a reduced number of macrophages were infected with mycobacteria consistent with TNF-{alpha} enhanced anti-mycobacterial activity in the cultures that received TNF-{alpha} (Fig. 1C) . However, IFN-{gamma} treatment offered less protective effect (Fig. 1D) , showing similar numbers of infected cells and intracellular microorganisms as the cultures that received M. avium alone (Fig. 1B) . These results suggest that a mechanism specific to IFN-{gamma} signaling is negatively impacted by M. avium.


Figure 1
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  		Figure 1. M. avium-infected macrophages are refractory to IFN-{gamma} activity. Macrophages were either left untreated (A and B) or were pretreated for 18 h with (C) TNF-{alpha} (10 ng/ml) or (D) IFN-{gamma} (10 ng/ml). Cells were then infected with M. avium (5:1) for 2 h (B–D), excess bacteria were removed, and cytokines were added at re-feeding time. By day 10, high levels of mycobacteria were observed in IFN-{gamma}-treated (D) and medium-treated cells (B), in contrast to reduced levels of bacteria TNF-{alpha} treated macrophages (C).

 
M. avium inhibits IFN-{gamma} signaling in macrophages
To study the mechanism responsible for the failure of IFN-{gamma} to promote mycobacterial clearance in treated macrophages, we evaluated the effect of M. avium infection on the IFN-{gamma} signaling cascade. Phosphorylation of STAT-1 has been documented as an early requirement for IFN-{gamma} downstream signaling events leading to STAT-1 dimerization and transcriptional activation of target genes. To determine whether mycobacteria reduce STAT-1 phosphorylation, macrophages were exposed to M. avium for 2 h, excess bacteria were removed, and cells were incubated for an additional 6 h to allow bacteria internalization prior to the addition of IFN-{gamma} to the cultures. Uninfected macrophages demonstrate rapidly enhanced phosphorylation of STAT-1 in response to IFN-{gamma} (Fig. 2 ). Whereas, infection with M. avium resulted in suppression of phosphorylation of STAT-1 (Fig. 2) as determined by Western blot and densitometric analysis, indicating that M. avium-induced suppression of IFN-{gamma} signaling involves down-regulation of the STAT signaling pathway. Analysis of cell surface IFN-{gamma} receptors shows no reduction in the expression of IFN-R1 chain in M. avium infected macrophages (data not shown), suggesting alternate mechanisms of reduced IFN signaling.


Figure 2
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  		Figure 2. M. avium inhibits IFN-{gamma}-induced signaling. Macrophages were exposed to M. avium (5:1) for 2 h, and non-adhered/non-internalized mycobacteria were removed by washing the cells twice with medium. After 8 h the cells were treated with IFN-{gamma} (10 ng/ml) for 30 min, and whole cell lysates were generated. STAT-1 was immunoprecipitated and analyzed by Western blotting for phospho-STAT-1. Equal protein loading was confirmed by stripping and re-probing the membrane with an antibody against STAT-1. M. avium-treated cells showed reduced STAT-1 phosphorylation in response to IFN-{gamma} (n=3). Inset: Representative donor shown. Data represent densitometric analysis of P-Stat-1 and Stat-1 expressed as fold increase in P-STAT-1 relative to control cells, mean ± SEM, (n=3) (*, P<0.05).

 
Enhanced SOCS gene expression in M. avium-infected macrophages
To further evaluate how early interactions of M. avium with the macrophage might lead to dysfunction in IFN-{gamma} responses and enhanced susceptibility to mycobacterial replication, we examined M. avium-induced signaling events, which may influence the ability of the cells to respond to IFN-{gamma}. It has been reported that SOCS can modulate the host response to IFN-{gamma} and prevent activation of STAT-1. Based on the evidence that M. avium inhibits IFN-{gamma} induced STAT-1 activation, we next investigated the effect of M. avium on gene expression for several members of the SOCS family. Analyses of macrophages from multiple donors clearly demonstrate increased transcriptional activity for SOCS-3 and SOCS-1 (Fig. 3A ). Furthermore, treatment of macrophages with mycobacterial LAM, a TLR2 agonist, triggered a similar pattern for SOCS gene expression. Densitometry analysis of the SOCS RPA shows 10–25 fold increase in SOCS-3 and SOCS-1 transcription in cells that were exposed to mycobacteria compared with control cells (Fig. 3B) . Similarly, LAM induced 8–25 fold enhancement in both SOCS-3 and SOCS-1, but not SOCS-5. Since SOCS-3 transcription was consistently up-regulated after M. avium infection, we focused on SOCS-3 protein expression. Western blot studies of M. avium-infected macrophages from three additional donors showed an increase in SOCS-3 protein expression after 8 h (Fig. 3C) , which was sustained 24 h after infection (data not shown), regardless of baseline expression of this protein. Furthermore, treatment of macrophage cultures with MAg, in parallel with intact bacteria, also resulted in enhanced SOCS-3 gene expression (Fig. 3D) .


Figure 3
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  		Figure 3. Mycobacteria-infected macrophages express increased levels of SOCS-3 and SOCS-1. (A) SOCS gene expression was analyzed by RPA after generating total mRNA from control, M. avium (5:1) and LAM (8 µg/ml) cells treated for 2 h (n=3). (B) Densitometric analysis confirmed the results obtained showing a rapid induction of SOCS-1 and SOCS-3, but not SOCS-5, after exposure to the M. avium and mycobacterial products (n=3). (C) Immunoprecipitation and Western blot analysis of M. avium-treated cells revealed an increase in SOCS-3 protein (representative donor shown). Densitometric analysis of SOCS-3 Western blot shows enhanced SOCS-3 expression mean ± SEM (n=3) (*, P ≤0.05). (D) Macrophage cultures treated with M. avium or MAg for 2 h show up-regulation of SOCS-3 gene expression as demonstrated by RT-PCR, mean ± SEM (n=3).

 
Effect of p38 MAPK inhibitor on M. avium SOCS-3 gene expression
p38 MAPK plays an important role in the innate and adaptive immune response, and several studies have implicated p38 MAPK as a signaling target for mycobacterial infections [32 , 33 ]. Exposure of human macrophages to M. avium results in an increase in the phosphorylation state of p38 MAPK, evident within minutes of M. avium interaction with macrophage hosts (Fig. 4A ). Incubation of the macrophages with M. avium antigen (MAg) (Fig. 3D ) shows similar results, albeit less dramatic. Because evidence supports a link between p38 MAPK, not only in the initiation of TLR2 signaling, but also in the induction of SOCS-3 gene expression [34 , 35 ], we examined whether obstruction of the p38 MAPK pathway would reduce M. avium-induced SOCS-3 transcription. Infected and control macrophages were incubated with or without the p38 MAPK inhibitor SB203580 at 10 µM for 60 min, exposed to M. avium (5:1) and SOCS-3 gene transcription examined by RPA. As shown in Fig. 4B and 4C , SB203580 reduced the ability of mycobacteria to induce SOCS-3 gene expression. These data correlated with down-regulated SOCS-3 protein as determined by immunoprecipitation from whole cell lysates (inset in Fig. 4C ). These results suggest that p38 MAPK represents a link in the pathway by which M. avium induces SOCS expression and blunts IFN responsiveness.


Figure 4
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  		Figure 4. M. avium induces p38 MAPK phosphorylation. (A) Human macrophages were incubated with M. avium or mycobacteria Ag (MAg) at 25 µg/ml for the indicated time periods. The cells were washed, and whole cell lysates were generated. The phosphorylation state of p38 MAPK was determined by Western blotting using an anti-phospho p38 antibody. Equal amount of protein loading was confirmed by stripping and re-probing the membrane with an antibody against p38 MAPK. M. avium-induced phoshorylation of p38 MAPK was evident after 5 min. (B) Macrophages were pretreated with SB203580 for 1 h prior to exposure to mycobacteria, mRNA generated and analyzed by RPA for SOCS gene expression, n = 2. (C) Densitometric analysis of RPA shown in (B). Inset: Representative Western blot showing reduced expression of SOCS-3 by p38 MAPK, n = 2.

 
Increased SOCS-3 protein expression in co-infected tissues
To extend our in vitro observations regarding IFN-{gamma} resistance, mediated via enhanced SOCS, we examined lymph nodes obtained from patients infected with both HIV and M. avium for SOCS protein expression. Whereas minimal immunohistochemical staining of SOCS protein was seen in the uninfected tissue (Fig. 5A ), with a modest increase in LN from HIV-positive-only subjects (Fig. 5B) , much higher SOCS-3 staining was apparent in those LN from M. avium and HIV-1 co-infected individuals (Fig. 5D and inset). Furthermore, the majority of cells expressing SOCS-3 protein were macrophages. Lymph node tissues from co-infected HIV and M. avium were negative when stained with isotype-matched control antibody (Fig. 5C) . Similar results were found when the co-infected lymph nodes were stained for SOCS-1 (data not shown). These data suggest a potential role for SOCS proteins in the pathogenesis of opportunistic infections in AIDS patients.


Figure 5
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  		Figure 5. SOCS-3 protein expression in macrophages of co-infected lymph node AIDS patients. (A) Tissues from HIV-negative LN, (B) HIV-positive LN, and (C, D) MAC and HIV co-infected HIV-positive individuals were analyzed for the presence of SOCS-3 protein by immunohistochemistry; n = 2. No staining was observed with an isotype-matched control antibody shown in (C) as negative control. The unstained filamentous structures in the cytoplasm of the positive cells (D) are consistent with mycobacteria. Inset: In situ HIV hybridization and M. avium detection in co-infected LN.

 
DISCUSSION

Evasion of host defense by M. avium in immune-compromised hosts reflects the lack of CD4+ T cells and their immunoregulatory cytokines, including IFN-{gamma}. However, therapeutic delivery of exogenous IFN-{gamma}, even with HAART treatment, is not always associated with resolution of local MAC lesions [36 ]. In this report, we provide further insight into the molecular mechanisms used by M. avium, which may ultimately favor mycobacterial survival within the macrophage. Our studies show that M. avium manipulation of IFN-{gamma} responses involves the induction of SOCS proteins—negative regulators of cytokine signaling—including IFN-{gamma}. Mycobacterial-induced SOCS transcription was associated with enhanced activation of p38 MAPK. Moreover, blockade of p38 MAPK signaling pathways resulted in reduced SOCS-3 gene and protein expression. We also present evidence showing that lymph nodes from co-infected AIDS patients possess increased levels of SOCS-3 in the macrophage population, consistent with resistance to anti-mycobacterial therapy.

Prior to the AIDS pandemic, M. avium diseases, typically the result of uncontrolled bacterial replication within macrophages, were unusual and occurred—with rare exceptions—only in cases associated with underlying malignancies and immune impairments such as inherited IFN-{gamma} abnormalities, defective STAT-1 responses, or therapeutic immunodeficiencies [14 , 16 , 37 38 39 40 ]. Individuals infected with HIV-1 are at most risk for disseminated mycobacterial disease with the vast majority of non-tuberculous infections in AIDS patients due to M. avium [2 , 3 ], but it can rarely be diagnosed in chronic pulmonary infections of normal hosts [41 ]. Although, since the advent of HAART, mortality due to M. avium has declined, infections of this nature can limit the quality of life and negatively affect clinical outcome of HIV-infected patients. In addition, immune reconstitution disease caused by M. avium, a manifestation of a restored immune response that develops transiently and shortly after HAART, has been increasingly reported in HIV-1 infected individuals [5 ].

IFN-{gamma}, an immunomodulator with known macrophage activating properties [42 43 44 ], reportedly has conflicting effects in resolution of both M. avium and M. tuberculosis infections within human monocyte/macrophages in vitro [18 , 45 46 47 48 49 50 ]. Results regarding the effect of IFN-{gamma} on mycobacterial infections have been reported, showing either a lack of IFN-{gamma} response, enhanced bacterial replication within macrophages, or at best modest anti-mycobacterial capacity. In the case of AIDS patients, susceptibility to OI has been attributed to the steady decline in CD4+ lymphocytes and as a consequence a lack in the production of IFN-{gamma}, especially at later stages of the disease. Despite the apparent complexity in defining the anti-mycobacterial effects of IFN-{gamma} in vitro, when IFN-{gamma} is used in conjunction with standard anti-mycobacterial and anti-retroviral therapy in patients with AIDS [36 , 47 , 51 52 53 ], either improved or limited enhanced immune responses against M. avium have been reported, although, in some cases, IFN-{gamma} therapy did not result in a prolonged benefit against M. avium [36 ].

In our studies, M. avium-infected macrophages become refractory to IFN-{gamma}, suggesting that M. avium modulates and activates pathways that interfere with interferon signaling. SOCS are a family of intracellular proteins, initially described to be induced by and that regulate responses to multiple cytokines, including IFN-{gamma} signaling [22 , 23 , 25 ]. Activation of p38 MAPK by TLR2 ligands, including mycobacteria, has been shown to facilitate mycobacteria replication and contribute to NF-{kappa}B activation [32 , 33 , 54 ]. We further implicate M. avium-induced p38 MAPK pathway as an important step for the increase in transcriptional regulation of SOCS in mycobacteria-infected macrophages as shown by the use of SB203580, a p38 MAPK inhibitor. In addition, p38 MAPK can potentially be involved in the enhanced expression of CCR5 and CXCR4 on the host cell induced by M. avium and M. tuberculosis [27 , 55 ]. SOCS-1 and SOCS-3 expression can be induced by various cytokines [25 , 33 , 56 ], and further studies are necessary to determine the contribution of mycobacteria-induced cytokines in the M. avium–induced SOCS expression. During a normal immune response, the induction of IFN-{gamma} SOCS expression may help control interferon actions to avoid uncontrolled inflammation. However, M. avium ingeniously manipulates this pathway to its benefit. Several studies have described the induction and participation of SOCS proteins in immune evasion by intracellular pathogens, including Listeria monocytogenes, Leishmania donovani, M. bovis, and Toxoplasma gondii [57 58 59 60 ]. Not only do we show that M. avium enhances SOCS-3 and SOCS-1 expression in vitro, molecules involved in the down-regulation of IFN-{gamma} responses [25 ], but we demonstrate increased SOCS-3 protein expression in co-infected LN of AIDS patients, consistent with a role for SOCS in connection with macrophage failure to clear mycobacteria in co-infected AIDS patients, which ultimately may favor both virus and bacteria survival. Although non-tuberculous infections in HIV seronegative patients are rare [61 ], preventing analysis of M. avium-only infected tissues for SOCS, our studies link co-infection with M. avium and HIV-1 with enhanced SOCS-3 expression in vivo, reflecting our in vitro studies.

The expression of these negative regulators of interferon signaling may potentially impede the anti-retroviral activity of type I and type II interferons by blocking interferon-induced genes, such as 2,-OAS and MxA [62 63 64 ]. SOCS exert their inhibitory effects through the interaction of the SH2 domain found in SOCS proteins with phosphotyrosines on cytokine receptors and signaling intermediates leading to blockade of JAK/STAT signaling. These negative regulators also contain a conserved C-terminal SOCS box that interacts with the E3-ligase complex leading to ubiquitination and subsequent proteaosomal degradation of STAT proteins [65 ]. Although initially the induction of SOCS was attributed to JAK-/STAT-dependent cytokines, TLR triggering ligands induce and are subject to regulation by SOCS family members [66 67 68 69 ]. The precise mechanism by which M. avium benefits from SOCS is still unclear, and it is not known whether SOCS induction by mycobacteria may target additional host molecules necessary for an effective immune response to proteasomal degradation. However, our study provides further understanding of how M. avium modulates the host immune response to its benefit and, as a consequence, may also contribute to successful HIV replication. Recent evidence implicates SOCS in the modulation/attenuation of HIV-1 immune responses [70 71 72 ]. The targeting of natural immune inhibitors, such as SOCS, has been suggested as an alternative approach to develop effective therapeutic HIV vaccines [70 ]. This could offer a dual advantage by controlling and improving the responses to IFN-{gamma} during mycobacterial infections while enhancing anti-retroviral immunity [70 ].

Our early studies show that a reciprocal relationship exists between HIV and M. avium, as demonstrated by increased viral replication in co-infected LN tissues, enhanced NF-{kappa}B activation leading to increased CCR5 cell surface expression on macrophages, and production of chemotactic factors [7 , 73 , 74 ]. Here, we describe SOCS proteins as mycobacterial allies in the conquest of the macrophage host and emphasize the role of p38 MAPK pathway in the successful survival of M. avium within the host. Overall, the present study provides evidence to support the manipulation of IFN-{gamma} signaling responses in macrophages by intracellular SOCS-3 and SOCS-1, in conjunction with early signaling events engaged by mycobacteria. It also emphasizes the importance of achieving the appropriate balance between M. avium and IFN-{gamma} signaling, presenting the possibility of targeting these negative regulators to improve interferon dependent immune responses and anti-mycobacterial therapy.

ACKNOWLEDGEMENTS

This work was supported by the Intramural Research Program of the NIH, National Institute of Dental and Craniofacial Research. The authors would like to thank Drs. Nancy McCartney-Francis, Niki Moutsoupolos, and Gikas Katsifis for their suggestions and constructive comments. We also thank Dr. Carl G. Feng (Laboratory of Parasitic Diseases, NIAID, NIH) for providing M. avium.

Received March 15, 2006; revised June 2, 2006; accepted June 5, 2006.

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