Increased STAT3 Activation in Periodontitis Drives Inflammatory Bone Loss.

Periodontitis is one of the most prevalent human inflammatory diseases. It is characterized by periodontal tissue destruction, progressively driven by the host response. In this regard, cytokines associated with tissue destruction, such as interleukin (IL)-6 and IL-23, use a common signaling pathway mediated by STAT3. This transcription factor is also needed for IL-17A production, a key mediator in periodontitis pathogenesis. Although several studies have reported increased activation of STAT3 in experimental periodontitis, a detailed characterization of STAT3 activation in human gingival tissues and its involvement in alveolar bone loss has yet to be explored. Using a cross-sectional study design, we detected increased proportions of pSTAT3-positive cells during periodontitis compared with health, particularly in epithelial cells and T cells. Other cell types of hematopoietic and nonhematopoietic origin also display STAT3 activation in gingival tissues. We detected increased STAT3 phosphorylation and expression of STAT3-related genes during experimental periodontitis. Next, we evaluated the role of STAT3 in alveolar bone destruction using a mouse model of STAT3 loss of function (mut-Stat3 mice). Compared with controls, mut-Stat3 mice had reduced alveolar bone loss following ligature-induced periodontitis. We also evaluated pharmacologic inhibition of STAT3 in ligature-induced periodontitis. Like mut-Stat3 mice, mice treated with STAT3 small-molecule inhibitor had reduced bone loss compared with controls. Our results demonstrate that STAT3 activation is increased in epithelial and T cells during periodontitis and indicate a pathogenic role of STAT3 in inflammatory alveolar bone loss.

Establishment and Stability of the Murine Oral Microbiome.

Commensal microbiomes exert critical functions at barrier sites. In particular, establishment of the commensal microbiome after birth dictates immune functionality and tissue homeostasis at mucosal surfaces. To investigate the establishment and stability of the oral mucosal microbiome in mice, we evaluated oral microbiome communities shortly after birth, through adulthood, and up to 1 y of life in a controlled manner, using sequential oral samples from the same mice over time. We further evaluated transmissibility of oral microbiomes from parents and during cohousing experiments and evaluated susceptibility to oral inflammatory disease in mice harboring distinct microbiomes. Our work reveals basic principles in the establishment and stability of a health-associated oral microbiome after birth and provides insights that may be important for host-microbiome experimentation in animal models.

Intestinal macrophages and response to microbial encroachment.

Macrophages in the gastrointestinal mucosa represent the largest pool of tissue macrophages in the body. In order to maintain mucosal homeostasis, resident intestinal macrophages uniquely do not express the lipopolysaccharide (LPS) co-receptor CD14 or the IgA (CD89) and IgG (CD16, 32, and 64) receptors, yet prominently display Toll-like receptors (TLRs) 3-9. Remarkably, intestinal macrophages also do not produce proinflammatory cytokines in response to TLR ligands, likely because of extracellular matrix (stromal) transforming growth factor-ß (TGF-ß) dysregulation of nuclear factor (NF)-κB signal proteins and, via Smad signaling, expression of IκBα, thereby inhibiting NF-κB-mediated activities. Thus, in noninflamed mucosa, resident macrophages are inflammation anergic but retain avid scavenger and host defense function, an ideal profile for macrophages in close proximity to gut microbiota. In the event of impaired epithelial integrity during intestinal infection or inflammation, however, blood monocytes also accumulate in the lamina propria and actively pursue invading microorganisms through uptake and degradation of the organism and release of inflammatory mediators. Consequently, resident intestinal macrophages are inflammation adverse, but when the need arises, they receive assistance from newly recruited circulating monocytes.

Differential mucosal susceptibility in HIV-1 transmission and infection.

HIV infection occurs primarily through mucosal surfaces, indicating that protection at mucosal sites may be crucial in prevention and treatment. The host innate and adaptive immune elements provide a level of protection, which differs between mucosal compartments, and appears to be most successful in the oral environment, where transmission is rare. In addition to the distinct oral mucosal architecture and cellular constituents, oral fluids, unlike other mucosal secretions, are rarely a vehicle for HIV infection. Multiple soluble factors may contribute to this antiviral activity, including neutralizing antibodies, secretory leukocyte protease inhibitor (SLPI), antiviral peptides such as defensins and cystatins, glycoproteins including thrombospondin and lactoferrin, and complement components. Understanding the antiviral activities of these and other potential resistance factors is becoming increasingly important in attempts to design treatments in the era of HAART resistance. In this regard, the mechanism of anti-HIV action of SLPI has recently been further elucidated by the discovery of its binding protein/receptor, which plays a key role in the infection of macrophages and may consequently be a novel therapeutic target. Continued elucidation of the unique features of mucosal HIV immunology is essential for understanding HIV pathogenesis and for developing effective vaccines and therapeutics.

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-beta in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.

Permissive factors for HIV-1 infection of macrophages.

Immunodeficiency, the consequence of HIV-1 infection, predisposes the host to opportunistic infections. In turn, opportunistic pathogens influence target cell susceptibility to HIV-1 infection and replication. Although the advent of highly active antiretroviral therapy (HAART) has altered these sequelae, co-infections may prevail in some parts of the world and in failed HAART regimens. Moreover, immune activation as occurs in tonsil and non-infectious mucosal inflammatory lesions may also be associated with proximal sites of viral replication. These connections between enhancement of HIV-1 infection and activation/inflammation warrant further elucidation of the factors promoting permissiveness to HIV-1 infection. Using the opportunistic pathogen Mycobacterium avium as an in vitro model, we demonstrated that co-infection facilitated HIV-1 infection of monocyte-macrophages by multiple pathways. M. avium activated NF-kappaB, the downstream consequences of which included augmented expression of tumor necrosis factor alpha and CCR5 receptors, both permissive for sustaining HIV-1 infection. Pronounced viral replication in lymph nodes co-infected with M. avium and HIV-1 paralleled these in vitro findings. Furthermore, reduction in viral burden is associated with treatment of infected or inflamed tissues, underscoring the link between immune activation and viral replication.

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-beta (TGF-beta), has made it possible to explore the contribution of the SMAD proteins to TGF-beta activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-beta 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-beta. 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.

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.

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.

Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression.

Infection with HIV-1 results in pronounced immune suppression and susceptibility to opportunistic infections (OI). Reciprocally, OI augment HIV-1 replication. As we have shown for Mycobacterium avium complex (MAC) and Pneumocystis carinii, macrophages infected with opportunistic pathogens and within lymphoid tissues containing OI, exhibit striking levels of viral replication. To explore potential underlying mechanisms for increased HIV-1 replication associated with coinfection, blood monocytes were exposed to MAC antigens (MAg) or viable MAC and their levels of tumor necrosis factor alpha (TNFalpha) and HIV-1 coreceptors monitored. MAC enhanced TNFalpha production in vitro, consistent with its expression in coinfected lymph nodes. Using a polyclonal antibody to the CCR5 coreceptor that mediates viral entry of macrophage tropic HIV-1, a subset of unstimulated monocytes was shown to be CCR5-positive by fluorescence-activated cell sorter analysis. After stimulation with MAg or infection with MAC, CCR5 expression was increased at both the mRNA level and on the cell surface. Up-regulation of CCR5 by MAC was not paralleled by an increase in the T cell tropic coreceptor, CXCR4. Increases in NF-kappaB, TNFalpha, and CCR5 were consistent with the enhanced production of HIV-1 in MAg-treated adherent macrophage cultures as measured by HIV-1 p24 levels. Increased CCR5 was also detected in coinfected lymph nodes as compared with tissues with only HIV-1. The increased production of TNFalpha, together with elevated expression of CCR5, provide potential mechanisms for enhanced infection and replication of HIV-1 by macrophages in OI-infected cells and tissues. Consequently, treating OI may inhibit not only the OI-induced pathology, but also limit the viral burden.