Pathogens are detected by innate immune receptors that, upon activation, orchestrate an appropriate immune response. Recent studies revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella abortus infection. However, no report has elucidated the role of inflammasome receptors in Brucella recognition. Therefore, we decided to investigate the function of NLRC4, NLRP3, and AIM2 in sensing Brucella. In this study, we showed that NLRC4 is not required to induce caspase-1 activation and further secretion of IL-1β by B. abortus in macrophages. In contrast, we determined that AIM2, which senses Brucella DNA, and NLRP3 are partially required for caspase-1 activation and IL-1β secretion. Additionally, mitochondrial reactive oxygen species induced by Brucella were implicated in IL-1β production. Furthermore, AIM2, NLRP3, ASC, and caspase-1 knockout mice were more susceptible to B. abortus infection than were wild-type animals, suggesting that multiple ASC-dependent inflammasomes contribute to host protection against infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than pyroptosis, because we observed augmented bacterial burden in IL-1R and IL-18 knockout mice. Finally, we determined that bacterial type IV secretion system VirB and live, but not heat-killed, Brucella are required for full inflammasome activation in macrophages during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.

c Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-␣), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1␤ (IL-1␤), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-␣ and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-␥) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival. Brucellosis is zoonotic disease caused by Brucella species, which is distributed worldwide and affects over 500,000 people annually (1) and for which there is no approved efficacious human vaccine available. Brucella melitensis, B. suis, and B. abortus are the most pathogenic species for humans and are responsible for the vast majority of human cases (2). The infection can be transmitted to humans by several ways, among which inhalation of infected aerosols is one of the most frequent. The easy aerosolization and airborne transmission of Brucella species has contributed to their consideration as potential biological weapons (1) and their classification by the CDC and NIAID as category B bioterrorism agents. Outbreaks of human brucellosis due to airborne transmission have been reported in different settings, including abattoirs, vaccine production laboratories, and rural areas (3-5). Notably, aerosols have been implicated in most cases of laboratory-acquired brucellosis, which is considered the commonest laboratory-acquired infection (6).Lung epithelial cells and alveolar macrophages (AM) are the first cells to be contacted by inhaled microorganisms. AM constitute the first line of pulmonary defense and are capable of initiating a local immune respons...

IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.

dBrucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-B signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-␣) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.

Brucella abortus is a Gram-negative bacterium that survives inside host cells as a facultative intracellular pathogen (1). It infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. In humans, brucellosis symptoms include undulant fever, endocarditis, arthritis, and osteomyelitis (2). In cattle, B. abortus causes miscarriage and infertility, leading to serious economic losses (3). The host immune response to B. abortus is initiated through innate immune mechanisms that recognize bacterial components and provide the necessary signals for the induction of an adaptive immune response (4). This specific adaptive immune response, which is Th1 mediated, plays a critical role in host control of B. abortus (5). The Th1-driven immune response to B. abortus involves CD4 ϩ and CD8 ϩ T lymphocytes, macrophages, dendritic cells (DCs), and proinflammatory cytokines such as gamma interferon (IFN-␥) and interleukin-12 (IL-12) (6-9).There is a growing interest in the immunoregulatory role of lipid mediators in infectious diseases (10-12). 5-Lipoxygenase (5-LO) is an enzyme required for the biosynthesis of two important groups of lipid mediators, leukotrienes (LTs) and lipoxins (LXs), both derived from arachidonic acid. 5-LO is made in several cell types, including neutrophils, eosinophils, monocytes/macrophages, dendritic cells, mast cells, and lymphocytes (13). To produce LTs, 5-LO acts on arachidonic acid, converting it to leukotriene A 4 (LTA 4 ). LTA 4 is then converted into leukotriene B 4 (LTB 4 ) or leukotriene C 4 (LTC 4 ), molecules that are exported from the cell (14). LTs are produced at the initial steps of the acute inflammatory response and act predominantly as proinflammatory lipid mediators. LTB 4 , for example, attracts neutrophils, monocytes, and lymphocytes to the site of inflammation and induces edema formation by increasing vascular permeability and plasma leakage at the site of inflammation (15-18). In contrast to LTs, production of LXs is dependent on cell-cell interaction by a process known as transcellular biosynthesis and requires the interaction between 5-LO and 15-or 12-lipoxygenase (15-LO or 12-LO, respectively) (19). Production of LXs by 5-15-LOs begins in eosinophils, monocytes, or epithelial cells (18). The 15-LO metabolic product, 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE), is released by these cells, taken up by polymorphonuclear cells or monocytes, and then processed by 5-LO into bioactive lipoxin A 4 (LXA 4 ) or lipoxin B 4 (LXB 4 ). Production of LXs by 15-12-LO occurs after arachidonic acid conversion to LTA 4 by 5-LO in leukocytes. LTA 4 is taken up by platelets and transformed to LXA 4 and LXB 4 via 12-LO activity (20). LXs act as anti-inflammatory and proresolution lipid mediators, by inhibiting both neutrophil and eosinophil transmigration into sites of infection and by promoting the noninflammatory infiltration of monocytes that is required for resolution and wound healing (21-23). LXs also stimulate macrophages to ingest and clear apoptotic neutrop...