Teresa Greenwell-Wild

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-β (TGF-β), has made it possible to explore the contribution of the SMAD proteins to TGF-β activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-β to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-β. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-microbial properties found in mucosal fluids. It is expressed during cutaneous wound healing. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in this process. We have generated mice null for the gene encoding SLPI (Slpi), which show impaired cutaneous wound healing with increased inflammation and elastase activity. The altered inflammatory profile involves enhanced activation of local TGF-β in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.

Topical Estrogen Accelerates Cutaneous Wound Healing in Aged Humans Associated with an Altered Inflammatory Response

The effects of intrinsic aging on the cutaneous wound healing process are profound, and the resulting acute and chronic wound morbidity imposes a substantial burden on health services. We have investigated the effects of topical estrogen on cutaneous wound healing in healthy elderly men and women, and related these effects to the inflammatory response and local elastase levels, an enzyme known to be up-regulated in impaired wound healing states. Eighteen health status-defined females (mean age, 74.4 years) and eighteen males (mean age, 70.7 years) were randomized in a double-blind study to either active estrogen patch or identical placebo patch attached for 24 hours to the upper inner arm, through which two 4-mm punch biopsies were made. The wounds were excised at either day 7 or day 80 post-wounding. Compared to placebo, estrogen treatment increased the extent of wound healing in both males and females with a decrease in wound size at day 7, increased collagen levels at both days 7 and 80, and increased day 7 fibronectin levels. In addition, estrogen enhanced the strength of day 80 wounds. Estrogen treatment was associated with a decrease in wound elastase levels secondary to reduced neutrophil numbers, and decreased fibronectin degradation. In vitro studies using isolated human neutrophils indicate that one mechanism underlying the altered inflammatory response involves both a direct inhibition of neutrophil chemotaxis by estrogen and an altered expression of neutrophil adhesion molecules. These data demonstrate that delays in wound healing in the elderly can be significantly diminished by topical estrogen in both male and female subjects.

Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti–HIV-1 activity

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytidine deaminase, is a recently recognized innate intracellular protein with lethal activity against human immunodeficiency virus (HIV). Packaged into progeny virions, APOBEC3G enzymatic activity leads to HIV DNA degradation. As a counterattack, HIV virion infectivity factor (Vif) targets APOBEC3G for proteasomal proteolysis to exclude it from budding virions. Based on the ability of APOBEC3G to antagonize HIV infection, considerable interest hinges on elucidating its mechanism(s) of regulation. In this study, we provide the first evidence that an innate, endogenous host defense factor has the potential to promote APOBEC3G and rebuke the virus-mediated attempt to control its cellular host. We identify interferon (IFN)-α as a potent inducer of APOBEC3G to override HIV Vif neutralization of APOBEC3 proteins that pose a threat to efficient macrophage HIV replication. Our data provide a new dimension by which IFN-α mediates its antiviral activity and suggest a means to render the host nonpermissive for viral replication.

Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

The distribution of secretory leukocyte protease inhibitor (SLPI) at entry portals indicates its involvement in defending the host from pathogens, consistent with the ability of SLPI to inhibit human immunodeficiency virus (HIV)-1 infection by an unknown mechanism. We now demonstrate that SLPI binds to the membrane of human macrophages through the phospholipid-binding protein, annexin II. Based on the recent identification of human cell membrane phosphatidylserine (PS) in the outer coat of HIV-1, we define a novel role for annexin II, a PS-binding moiety, as a cellular cofactor supporting macrophage HIV-1 infection. Moreover, this HIV-1 PS interaction with annexin II can be disrupted by SLPI or other annexin II–specific inhibitors. The PS–annexin II connection may represent a new target to prevent HIV-1 infection.

Human Immunodeficiency Virus Type 1-Induced Macrophage Gene Expression Includes the p21 Gene, a Target for Viral Regulation

ABSTRACT In contrast to CD4+ T cells, human immunodeficiency virus type 1 (HIV-1)-infected macrophages typically resist cell death, support viral replication, and consequently, may facilitate HIV-1 transmission. To elucidate how the virus commandeers the macrophages intracellular machinery for its benefit, we analyzed HIV-1-infected human macrophages for virus-induced gene transcription by using multiple parameters, including cDNA expression arrays. HIV-1 infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis, and the cell cycle, among which the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) gene was the most prominent. p21 mRNA and protein expression followed a bimodal pattern which was initially evident during the early stages of infection, and maximum levels occurred concomitant with active HIV-1 replication. Mechanistically, viral protein R (Vpr) independently regulates p21 expression, consistent with the reduced viral replication and lack of p21 upregulation by a Vpr-negative virus. Moreover, the treatment of macrophages with p21 antisense oligonucleotides or small interfering RNAs reduced HIV-1 infection. In addition, the synthetic triterpenoid and peroxisome proliferator-activated receptor γ ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which is known to influence p21 expression, suppressed viral replication. These data implicate p21 as a pivotal macrophage facilitator of the viral life cycle. Moreover, regulators of p21, such as CDDO, may provide an interventional approach to modulate HIV-1 replication.

Interleukin-27 inhibition of HIV-1 involves an intermediate induction of type I interferon

Infection of CD4(+) chemokine coreceptor(+) targets by HIV is aided and abetted by the proficiency of HIV in eliminating or neutralizing host cell-derived defensive molecules. Among these innate protective molecules, a family of intracellular apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases, is constitutively expressed but inactivated by HIV viral infectivity factor. The ability of interferon-alpha (IFN-alpha) to augment cytidine deaminases offered the possibility that the balance between virus and target cell might be altered in favor of the host. Further characterization of transcriptional profiles induced by IFN-alpha using microarrays, with the intention to identify and dissociate retroviral countermaneuvers from associated toxicities, revealed multiple molecules with suspected antiviral activity, including IL-27. To establish whether IFN-alpha toxicity might be sidestepped through the use of downstream IL-27 against HIV, we examined whether IL-27 directly regulated cytidine deaminases. Although IL-27 induces APOBECs, it does so in a delayed fashion. Dissecting the underlying regulatory events uncovered an initial IL-27-dependent induction of IFN-alpha and/or IFN-beta, which in turn, induces APOBEC3, inhibited by IFN-alpha/beta receptor blockade. In addition to macrophages, the IL-27-IFN-alpha connection is operative in CD4(+) T cells, consistent with an IFN-alpha-dependent pathway underlying host cell defense to HIV.

Viral and host cofactors facilitate HIV-1 replication in macrophages.

Human immunodeficiency virus type 1 (HIV‐1) infection of CD4+ T lymphocytes leads to their progressive loss, whereas HIV‐1‐infected macrophages appear to resist HIV‐1‐mediated apoptotic death. The differential response of these two host‐cell populations may be critical in the development of immunodeficiency and long‐term persistence of the virus. Multiple contributing factors may favor the macrophage as a resilient host, not only supporting infection by HIV‐1 but also promoting replication and persistence of this member of the lentivirus subfamily of primate retroviruses. An encounter between macrophages and R5 virus engages a signal cascade eventuating in transcriptional regulation of multiple genes including those associated with host defense, cell cycle, nuclear factor‐κB regulation, and apoptosis. It is important that enhanced gene expression is transient, declining to near control levels, and during this quiescent state, the virus continues its life cycle unimpeded. However, when viral replication becomes prominent, an increase in host genes again occurs under the orchestration of viral gene products. This biphasic host response must fulfill the needs of the parasitic virus as viral replication activity occurs and leads to intracellular and cell surface‐associated viral budding. Inroads into understanding how HIV‐1 co‐opts host factors to generate a permissive environment for viral replication and transmission to new viral hosts may provide opportunities for targeted interruption of this lethal process.

Mycobacterium avium‐induced SOCS contributes to resistance to IFN‐γ‐mediated mycobactericidal activity in human macrophages

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‐γ plays an important role in protective immunity against bacterial infections, IFN‐γ responses are compromised in AIDS patients and evidence suggests that exogenous IFN‐γ is inadequate to clear the mycobacteria. To determine the mechanism by which M. avium survives intracellularly, even in the presence of IFN‐γ, we studied the effect of mycobacteria infection in macrophages during early IFN‐γ signaling events. M. avium infected cells exhibited a reduced response to IFN‐γ, 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‐γ, 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.

Co-Infection with Opportunistic Pathogens Promotes Human Immunodeficiency Virus Type 1 Infection in Macrophages

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on susceptible host cells that express both CD4 and chemokine co-receptors. The co-receptor CCR5 is associated with primary infection by macrophage-tropic virus isolates, whereas CXCR4 is commonly associated with T cell- and dual-tropic viruses. Once infected, lymphocytes and macrophages may replicate HIV-1 or harbor latent virus, depending on environmental factors and cellular activation. Immune activation is often associated with viremia, which is consistent with enhanced infection and viral replication in activated cells harboring virus. In this regard, opportunistic infections activate the immune system with the detrimental sequelae of enhanced viral replication and viremia. Under these conditions, viral expansion extends beyond T cells to tissue macrophages, many of which are co-infected with opportunistic pathogens. The opportunistic infections promote macrophage susceptibility to HIV-1 through cytokine modulation and altered chemokine co-receptors, potential targets for intervention.