The innate immune response in human tuberculosis is not completely understood. To improve our knowledge regarding the role of cathelicidin hCAP-18/LL37 in the innate immune response to tuberculosis infection, we used immunohistochemistry, immunoelectron microscopy, and gene expression to study the induction and production of the antimicrobial peptide in A549 epithelial cells, alveolar macrophages (AM), neutrophils, and monocyte-derived macrophages (MDM) after infection with Mycobacterium tuberculosis. We demonstrated that mycobacterial infection induced the expression and production of LL-37 in all cells studied, with AM being the most efficient. We did not detect peptide expression in tuberculous granulomas, suggesting that LL-37 participates only during early infection. Through the study of Toll-like receptors (TLR) in MDM, we showed that LL-37 can be induced by stimulation through TLR-2, TLR-4, and TLR-9. This last TLR was strongly stimulated by M. tuberculosis DNA. We concluded that LL-37 may have an important role in the innate immune response against M. tuberculosis.

BackgroundCD33 is a membrane receptor containing a lectin domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. CD33 is expressed by monocytes, and reduced expression of CD33 correlates with augmented production of inflammatory cytokines, such as IL-1β, TNF-α, and IL-8. However, the role of CD33 in the inflammation associated with hyperglycemia and diabetes is unknown. Therefore, we studied CD33 expression and inflammatory cytokine secretion in freshly isolated monocytes from patients with type 2 diabetes. To evaluate the effects of hyperglycemia, monocytes from healthy donors were cultured with different glucose concentrations (15-50 mmol/l D-glucose), and CD33 expression and inflammatory cytokine production were assessed. The expression of suppressor of cytokine signaling protein-3 (SOCS-3) and the generation of reactive oxygen species (ROS) were also evaluated to address the cellular mechanisms involved in the down-regulation of CD33.ResultsCD33 expression was significantly decreased in monocytes from patients with type 2 diabetes, and higher levels of TNF-α, IL-8 and IL-12p70 were detected in the plasma of patients compared to healthy donors. Under high glucose conditions, CD33 protein and mRNA expression was significantly decreased, whereas spontaneous TNF-α secretion and SOCS-3 mRNA expression were increased in monocytes from healthy donors. Furthermore, the down-regulation of CD33 and increase in TNF-α production were prevented when monocytes were treated with the antioxidant α-tocopherol and cultured under high glucose conditions.ConclusionOur results suggest that hyperglycemia down-regulates CD33 expression and triggers the spontaneous secretion of TNF-α by peripheral monocytes. This phenomenon involves the generation of ROS and the up-regulation of SOCS-3. These observations support the importance of blood glucose control for maintaining innate immune function and suggest the participation of CD33 in the inflammatory profile associated with type 2 diabetes.

Responses to mycobacterial and nonmycobacterial antigens were examined in bronchoalveolar cells (BAC) and peripheral blood mononuclear cells (PBMC) from patients with active pulmonary tuberculosis (n=16) and healthy subjects (n=23). DNA synthesis in BAC (but not PBMC) from tuberculosis patients was significantly increased in response to the mycobacterial antigens purified protein derivative (PPD), antigen 85, and mannose-capped lipoarabinomannan but not to nonmycobacterial antigens. The response to PPD was also increased in enriched alveolar lymphocytes from tuberculosis patients (P<.05). The frequency of interferon-gamma but not interleukin-4- or -10-producing cells by ELISAspot was increased in PPD-stimulated BAC from patients with tuberculosis (P<.05). Accessory function of alveolar macrophages for T lymphocyte responses was similar and suppressive activity was variably decreased in tuberculosis patients. Thus, there is compartmentalization of mycobacterial antigen-specific lymphocytes to the lungs during active tuberculosis that on challenge produce a Th1-type cytokine host response.

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Experimental studies of granuloma formation and antimycobacterial immunity suggest that the lung is uniquely susceptible to Mycobacterium tuberculosis infection. Growth of virulent M. tuberculosis strains and avirulent strains of Mycobacterium bovis BCG is more rapid and destructive in the lung compared to other organs, and far fewer organisms are needed for infection when bacteria are delivered by aerosol compared to intravenous infection (9). Thus, infection of the lung is an important factor affecting the growth and survival of mycobacteria (4). The inhalation of aerosol droplets containing bacilli leads to the deposition of bacilli in both conducting and distal airways where many organisms are removed by mucociliary mechanisms. However, in alveolar spaces resident macrophages phagocytose organisms, resulting in the expression of reactive oxygen and nitrogen radicals and multiple chemokines and cytokines (3,38,39,45,50,52). These innate immune responses do not control the early growth of either virulent M. tuberculosis or avirulent M. bovis BCG in murine lungs. Ultimately, control of both M. tuberculosis and M. bovis BCG infection is dependent on the recruitment and activation of major histocompatibility complex (MHC)-restricted CD4 ϩ and CD8ϩ T cells (6, 9, 11, 21, 33, 52). Whether permissive growth of M. tuberculosis or M. bovis BCG in alveolar spaces is due to an inability of alveolar macrophages to activate T cells, to the capacity of mycobacteria to impair alveolar macrophage function, or to the effects of alveolar protein factors on alveolar macrophage and T-cell function is not known. Recent studies have shown that the 19-kDa lipoprotein which is expressed by both M. bovis BCG and M. tuberculosis can inhibit MHC-II antigen processing and presentation in bone marrow-derived macrophages (31,32,34,47). The inhibition occurs by blocking gamma interferon (IFN-␥) signaling through a Toll-like receptor 2 (TLR-2)-dependent mechanism. The present study was undertaken to determine the ability of alveolar macrophages to serve as antigen-presenting cells (APC) and whether alveolar macrophage function is affected by mycobacterial infection and exposure to mycobacterial lipoproteins. We determined that resident murine alveolar macrophages have the necessary surface molecules to serve as efficient APC for CD4 ϩ T cells, that they can process and present MHC-II-restricted antigens, and that the APC function of alveolar macrophages was inhibited by M. bovis BCG infection and the 19-kDa lipoprotein. Thus, the mycobacterial infection of alveolar macrophages may impede activation of CD4 ϩ T cells and contribute to the permissive pulmonary microenvironment supporting mycobacterial growth and persistence. MATERIALS AND METHODSMice. Specific pathogen-free, female C57BL/6 (H-2 b ) mice were purchased from Charles River Laboratories (North Wilmington, Mass.) and used at between 8 and 12 weeks of age. TLR-2 gene knockout mice were generously provided by O. Takeuchi and S. Akira (Osaka University, Osaka, Japan) and bred onto the...