Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections

Abstract

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral routes for means of vaccination.

N -Formyl-Perosamine Surface Homopolysaccharides Hinder the Recognition of Brucella abortus by Mouse Neutrophils

“…Indeed, Brucellainfected PMNs do not degranulate and induce low levels of reactive oxygen species (ROS) and cytokines, and the infection follows its course without significant neutrophilia (5,7,8). Likewise, the PMNs infiltration of the cervical lymph nodes after oral infection is very low, even in well-developed granulomas after 15 days of infection (9). Moreover, after 5 days of infection, the bacterium is found within M and DCs of mice but seldom inside PMNs in the target organs (10).…”

COX-2 Inhibition Reduces Brucella Bacterial Burden in Draining Lymph Nodes

“…We have first compared three different Brucella strains ( B. abortus 2308, B. melitensis 16M, and B. suis 1330) and their ability to colonize CLN at 8 and 15 days post-infection using the oral infection model we previously shown to induce a specific colonization of CLN (von Bargen et al, 2014). We avoided to use intraperitoneal inoculation since it is associated to systemic infection and recedes from Brucella physiological routes of infection or vaccination (Grilló et al, 2012).…”

“…WT C57BL/6 mice were inoculated with B. melitensis by oral administration (with 10 9 bacteria per mouse) as previously described (von Bargen et al, 2014) or by intranasal (with 10 5 bacteria per mice), conjunctival (10 9 bacteria per mice) or intradermal (10 4 bacteria per mice) routes. Mice were sacrificed at 8 and 29 days post-infection and the weight and bacterial load of CLN and spleens were measured ( Figures 3A,B ).…”

“…We also included the intradermal infection route often used for vaccination (Kim et al, 2012). We focused on CLN since CLN also drains the eye (Hamrah and Dana, 2007) and the nasal mucosa (Wolvers et al, 1999) and we previously shown that this organ was preferentially targeted by Brucella after oral inoculation (von Bargen et al, 2014). COX-2 being important during inflammatory process, contrary to the in vitro experiments, we decided to use B. melitensis as it seems that this species induced an enhanced inflammation in CLN compared to B. abortus .…”

“…Inocula were prepared by pelleting bacteria and by adjusting bacterial concentration in endotoxin free PBS (Gibco) to obtain the right concentration depending on the inoculation route. Oral infection was performed as previously described (von Bargen et al, 2014). Briefly, pelleted bacteria were re-suspended to previously heat-sterilized 5% milk and adjusted at respective MOI in 5% milk (Scharlau).…”