Mtb may block the recruitment of iNOS to the phagosomal membrane, avoiding exposure to NO because of distinct subcellular localization (Davis et al., 2007). In addition, Mtb may increase the expression

of Arg1, leading to competition with iNOS for its substrate (l-arginine) and drastic reduction in NO production (El Kasmi et al., 2008; Qualls et al., 2010). Evidence for this alternative mechanism was observed in mice models in which Arg1-deficient macrophages produced higher levels of NO, which contributed to Mtb intracellular death (El Kasmi et al., 2008). Although the Arg1–iNOS competition mechanism is well documented in murine models, little is known about how Mtb-infected human macrophages Fludarabine supplier respond to infection. It has been proposed that cultured human macrophages do not express either Arg1 or iNOS and do not produce NO (Fang & this website Nathan, 2007). However, findings based on cultured human macrophages may not reproduce the complexity of Mtb infection of human lung in vivo. Consistently, expression of iNOS and NO was reported in Mtb-infected human tissues (Choi et al., 2002). In this work, we investigated the expression of Arg1 in human tissues from patients with TB. Our findings show that Arg1 is produced in granuloma-associated macrophages and type II pneumocytes, but

not in lymphocytes. Paraffin-embedded human lung tissue biopsies, previously obtained for diagnostic purposes, were used in this study with the approval of Ethics Committee of the University of the State of Rio de Janeiro (protocol no.: 0034.0.325.000-10). Lung tissues were obtained from five patients with TB, all HIV negative, who underwent pulmonary resection. For controls, lung tissues from five randomly chosen individuals who had undergone resectional surgery for necropsy examination were used. Sections of paraffin-embedded human lungs were deparaffinized in xylene, hydrated, and treated with 10 mM citrate buffer (pH 6.2) at 95–98 °C for 20 min.

Subsequently, the sections were blocked with free serum for 15 min and treated with 3% H2O2 in PBS for 8 min at room temperature, rinsed with Tris buffer 0.05 M (pH 7.4) and incubated overnight at 4 °C with monoclonal anti-Arg1 (BD Biosciences) at 1 : 1000 in Tris buffer containing 1% bovine Sodium butyrate albumin. The anti-Arg1 antibody used here has previously been shown to be highly specific for Arg1 (El Kasmi et al., 2008). Expression of arginase 2 (Arg2) and iNOS was studied using polyclonal antibodies (Santa Cruz; 1 : 500 dilution). Secondary antibodies were incubated for 15 min at room temperature. Sections were immunostained with a biotin-free MACH 4™ Universal HRP-Polymer Detection Kit (Biocare Medical). The reaction was developed using the DAB Chromogen Kit (Biocare Medical). Sections were also stained with hematoxylin and eosin (HE).