Ex Vivo Differential Responsiveness to Clostridium perfringens and Lactococcus lactis by Avian Small Intestine Macrophages and T Cells

Abstract

Tissue resident immune system cells in the chicken intestine play a significant role in the protection against pathogens. However, very little is known about these cells. The current study was conducted to further characterize chicken intestinal immune system cells. Furthermore, this study aimed to assess the immune modulatory action of a highly virulent Clostridium perfringens, a commonly found chicken intestinal microbe, in comparison with a non-commensal, Lactococcus lactis, on intestine-derived immune system cells. The results demonstrated varying distribution of innate and adaptive immune cells along the avian gut-associated lymphoid tissue (GALT) in the duodenum, jejunum, ileum, and cecal tonsils. In addition, steady-state and tissue-specific presence of CD25+ cells among αβ and γδ T-cell subsets was assessed along the intestine. Ex vivo stimulation with C. perfringens or L. lactis resulted in a significant increase in the frequency of CD25+ T cells (γδ and αβ T cells). In addition, significantly more cell death was observed in ex vivo stimulation with C. perfringens, which was indirectly correlated with a decrease in macrophage activation based on nitric oxide (NO) production with no effect on lymphoid cell responsiveness as per intracellular interferon (IFN)-gamma (γ) staining. Ex vivo stimulation with L. lactis activated γδ T cells and αβ T cells, based on intracellular IFN-γ staining, while it had limited effect on macrophages. However, the ability of γδ and αβ T cells to produce IFN-γ and the ability of macrophages production of NO was rescued in the presence of L. lactis. These results demonstrate the potential application of L. lactis, as a probiotic, against virulent C. perfringens infection in chicken.

Cellular Immune Responses in Lymphoid Tissues of Broiler Chickens Experimentally Infected with Necrotic Enteritis–Producing Clostridium perfringens Strains

“…Previous work, albeit using in vitro and ex vivo experiments, has shown that virulent CP can activate chicken macrophages, resulting in an increased frequency of these cells (22, 38, 39). Another study also found that an ex vivo stimulation of chicken intestine–derived lymphocytes with a virulent CP strain (CP4) led to an activation of γδ T-cells, as determined by an upregulated surface expression of CD25 and intracellular IFNγ production in these cells (21). To this end, in the present study, the frequency of γδ T-cells in the CT of CP26-infected chickens was also found increased, suggesting involvement in the host response during NE.…”

“…Our previous work has shown that protection against NE is associated with secretory products from virulent, but not avirulent, CP (14) and identified certain secreted proteins as potential vaccine candidates (7,15,16). Studies aimed at characterizing CP-induced immune responses have shown elevated intestinal gene expression of antimicrobial peptides and cytokines such as interleukin (IL)-1b, IL-6, IL-8, IL-10, IL-12, and transforming growth factor b in NEaffected chickens (17,18,19). Our recent studies also found that broiler chickens experimentally infected with virulent CP had increased expression of proinflammatory cytokine genes IL-1b, IL-6, or interferon (IFN)c, in the intestinal and cecal tonsil (CT) tissues (13,20).…”

“…Although gene expression analysis can provide clues about molecular mechanisms of host-CP interactions, it is also critically important to understand the dynamics of these interactions at a cellular level. Our recent in vitro and ex vivo studies showed that stimulation of CT cells, splenocytes, and cells derived from intestinal lymphoid tissues with virulent CP can augment macrophage, γδ, CD4 + , and CD8 + T-cell frequencies, as well as activation status (20, 21, 22). For example, in vitro stimulation of a chicken macrophage cell line (MQ-NCSU) with an avirulent strain (CP5) and virulent strains (CP1 and CP26) showed that the virulent strains can activate macrophages and the production of proinflammatory cytokines (IL-1, IL-6, and IFNγ), as well as augment surface expression of MHC-II antigen–presenting molecule (22).…”

“…Furthermore, ex vivo stimulation primary cells from chicken spleen and CTs with CP26 led to increased CD4 + and CD8 + T-cell frequencies (20). In another ex vivo study, we found that stimulation of cells derived from intestinal lymphoid tissues with a virulent CP strain (CP4) resulted in increased cellular frequencies of macrophages, γδ T-cells, CD4 + , and CD8 + T-cells, including the surface expression of CD25 and intracellular production of IFNγ (21). However, lymphoid cellular responses in chickens against CP strains varying in in vivo virulence are yet poorly understood.…”

Intracloacal Inoculation of Broiler Chickens with Clostridium perfringens Strains: Evaluation of Necrotic Enteritis Disease Development and Lymphoid Immune Responses

“…For example, broiler chickens coinfected with E. maxima and virulent C. perfringens induced an increased intestinal gene expression of pro-inflammatory cytokines (IL-1β, IFNγ, IL-17, IL-8, and others) [ 16 , 36 ], while in vitro stimulation of chicken intestinal epithelial cells with virulent C. perfringens or purified alpha-toxin led to an increased transcription of IL-6, IL-8, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF), and nuclear factor kappa-B (NFkB) genes [ 37 ]. In support of these findings, macrophages have been shown as the likely source of such mucosal inflammatory responses [ 18 , 38 ] [Kulkarni, et. al., 2023; unpublished].…”