Matthew C. Little

The Characterization of Intraepithelial Lymphocytes, Lamina Propria Leukocytes, and Isolated Lymphoid Follicles in the Large Intestine of Mice Infected with the Intestinal Nematode Parasite Trichuris muris

Despite a growing understanding of the role of cytokines in immunity to the parasitic helminth Trichuris muris, the local effector mechanism culminating in the expulsion of worms from the large intestine is not known. We used flow cytometry and immunohistochemistry to characterize the phenotype of large intestinal intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) from resistant and susceptible strains of mouse infected with T. muris. Leukocytes accumulated in the epithelium and lamina propria after infection, revealing marked differences between the different strains of mouse. In resistant mice, which mount a Th2 response, the number of infiltrating CD4+, CD8+, B220+, and F4/80+ IEL and LPL was generally highest around the time of worm expulsion from the gut, at which point the inflammation was dominated by CD4+ IEL and F4/80+ LPL. In contrast, in susceptible mice, which mount a Th1 response, the number of IEL and LPL increased more gradually and was highest after a chronic infection had developed. At this point, CD8+ IEL and F4/80+ LPL were predominant. Therefore, this study reveals the local immune responses underlying the expulsion of worms or the persistence of a chronic infection in resistant and susceptible strains of mouse, respectively. In addition, for the first time, we illustrate isolated lymphoid follicles in the large intestine, consisting of B cells interspersed with CD4+ T cells and having a central zone of rapidly proliferating cells. Furthermore, we demonstrate the organogenesis of these structures in response to T. muris infection.

Absence of CC Chemokine Ligand 2 Results in an Altered Th1/Th2 Cytokine Balance and Failure to Expel Trichuris muris Infection

Despite a growing understanding of the role of cytokines in immunity to intestinal helminth infections, the importance of chemokines has been neglected. As a chemokine with both chemoattractive properties and an ability to shape the quality of the adaptive immune response, CC chemokine ligand 2 (CCL2) was investigated as an attractive candidate for controlling resistance to these types of infection, which require highly polarized Th cell responses. We show here for the first time that CCL2 plays an important role in the development of resistance to infection by the gastrointestinal nematode Trichuris muris. Thus, in the absence of CCL2, worm expulsion does not occur, and the lymph node draining the site of infection becomes a Th1-promoting environment. Elevated levels of IL-12 are produced by polarizing APCs, and the composition of the APC environment itself is perturbed, with reduced numbers of macrophages.

Rapid dendritic cell mobilization to the large intestinal epithelium is associated with resistance to Trichuris muris infection.

The large intestine is a major site of infection and disease, yet little is known about how immunity is initiated within this site and the role of dendritic cells (DCs) in this process. We used the well-established model of Trichuris muris infection to investigate the innate response of colonic DCs in mice that are inherently resistant or susceptible to infection. One day postinfection, there was a significant increase in the number of immature colonic DCs in resistant but not susceptible mice. This increase was sustained at day 7 postinfection in resistant mice when the majority of the DCs were mature. There was no increase in DC numbers in susceptible mice until day 13 postinfection. In resistant mice, most colonic DCs were located in or adjacent to the epithelium postinfection. There were also marked differences in the expression of colonic epithelial chemokines in resistant mice and susceptible mice. Resistant mice had significantly increased levels of epithelium-derived CCL2, CCL3, CCL5, and CCL20 compared with susceptible mice. Furthermore, administering neutralizing CCL5 and CCL20 Abs to resistant mice prevented DC recruitment. This study provides clear evidence of differences in the kinetics of DC responses in hosts inherently resistant and susceptible to infection. DC responses in the colon correlate with resistance to infection. Differences in the production of DC chemotactic chemokines by colonic epithelial cells in response to infection in resistant vs susceptible mice may explain the different kinetics of the DC response.

Accumulation of eosinophils in intestine-draining mesenteric lymph nodes occurs after Trichuris muris infection

Eosinophils have recently been demonstrated capable of localizing to lymph nodes that drain mucosal surfaces, in particular during T helper 2 (Th2) responses. Resistance of mice to infection with the gastrointestinal nematode Trichuris muris depends critically on mounting of a Th2 response and represents a useful model system to investigate Th2 responses. Following infection of resistant BALB/c mice with T. muris, we observed accumulation of eosinophils in intestine‐draining mesenteric lymph nodes (MLNs). The accumulation of MLN eosinophils was initiated during the second week of infection and peaked during worm expulsion. In contrast, we detected a comparably late and modest increase in eosinophil numbers in the MLNs of infected susceptible AKR mice. MLN eosinophils localized preferentially to the medullary region of the lymph node, displayed an activated phenotype and contributed to the interleukin‐4 (IL‐4) response in the MLN. Despite this, mice genetically deficient in eosinophils efficiently generated IL‐4‐expressing CD4+ T cells, produced Th2 cytokines and mediated worm expulsion during primary T. muris infection. Thus, IL‐4‐expressing eosinophils accumulate in MLNs of T. muris‐infected BALB/c mice but are dispensable for worm expulsion and generation of Th2 responses, suggesting a distinct or subtle role of MLN eosinophils in the immune response to T. muris infection.

Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

Macrophages (Mφs) accumulate at sites of inflammation, and, because they can assume several functionally distinct states of activation, they can either drive or restrain inflammatory responses. Once believed to depend on the recruitment of blood monocytes, it is now clear that the accumulation of Mφs in some tissues can result from the proliferation of resident Mφs in situ. However, little is known about the proliferation and activation state of Mφ subsets in the gut during the development and resolution of intestinal inflammation. We show that inflammatory Mφs accumulate in the large intestine of mice during the local inflammatory response to infection with the gastrointestinal nematode parasite Trichuris muris. Classically activated Mφs predominate initially (as the inflammation develops) and then, following worm expulsion (as the inflammation resolves), both the resident and inflammatory populations of Mφs become alternatively activated. A small but significant increase in the proliferation of inflammatory Mφs is seen but only during the resolution phase of the inflammatory response following both worm expulsion and the peak in Mφ accumulation. In contrast to recent studies in the pleural and peritoneal cavities, the proliferation of resident and alternatively activated Mφs does not increase during the inflammatory response. Furthermore, in CCR2−/− mice, monocyte recruitment to the gut is impeded, and the accumulation of alternatively activated Mφs is greatly reduced. In conclusion, the recruitment of blood monocytes is the principle mechanism of Mφ accumulation in the large intestine. This study provides a novel insight into the phenotype and behavior of intestinal Mφ during infection-driven inflammation.

Characterisation of the protective immune response following subcutaneous vaccination of susceptible mice against Trichuris muris.

Graphical abstract

The role of CD8+ cells in the establishment and maintenance of a Trichuris muris infection

Chronic infection by the caecal‐dwelling intestinal murine nematode Trichuris muris occurs if given as a high‐dose infection to ‘susceptible’ AKR mice or as a low‐dose infection to the normally ‘resistant’ C57BL/6 mouse strain. Both regimes result in a type 1 cytokine response, i.e. high levels of IFN‐γ and IL‐12. Here we show this susceptible response is associated with a large population of CD8+ IFN‐γ+ cells within the mesenteric lymph nodes and numerous CD8+ cells infiltrating the caecal mucosa. Despite this, the in vivo abolition of CD8+ cells within AKR and C57BL/6 mice, either prior to infection or once infection has become established, failed to affect chronicity, implying that CD8+ T cells are not essential for the initiation or maintenance of the susceptible response to T. muris. Interestingly, the percentage of IFN‐γ+ CD4+ cells increased in treated groups, perhaps in a compensatory role. The majority of antigen‐specific cytokine responses were comparable in both treated and control groups, although IL‐5 was fivefold higher in animals receiving anti‐CD8 mAbs and IFN‐γ was also raised in treated mice. Mastocytosis was unaltered by CD8 depletion, however, paradoxically, eosinophilia within the caecum was reduced in treated mice. Together these data clearly demonstrate that CD8+ T cells are associated with chronic T. muris infection; however, these cells are dispensable for both the early and late phases of this response, but do appear to play a role in the regulation of certain cytokines and caecal eosinophilia.

Anti-inflammatory effects of infliximab in mice are independent of tumour necrosis factor α neutralization

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated ex vivo using enzyme‐linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

The Retinoic Acid Receptor Agonist Am80 Increases Mucosal Inflammation in an IL-6 Dependent Manner During Trichuris muris Infection

PurposeVitamin A metabolites, such as all-trans-retinoic acid (RA) that act through the nuclear receptor; retinoic acid receptor (RAR), have been shown to polarise T cells towards Th2, and to be important in resistance to helminth infections. Co-incidentally, people harbouring intestinal parasites are often supplemented with vitamin A, as both vitamin A deficiency and parasite infections often occur in the same regions of the globe. However, the impact of vitamin A supplementation on gut inflammation caused by intestinal parasites is not yet completely understood.MethodsHere, we use Trichuris muris, a helminth parasite that buries into the large intestine of mice causing mucosal inflammation, as a model of both human Trichuriasis and IBD, treat with an RARα/β agonist (Am80) and quantify the ensuing pathological changes in the gut.ResultsCritically, we show, for the first time, that rather than playing an anti-inflammatory role, Am80 actually exacerbates helminth-driven inflammation, demonstrated by an increased colonic crypt length and a significant CD4+ T cell infiltrate. Further, we established that the Am80-driven crypt hyperplasia and CD4+ T cell infiltrate were dependent on IL-6, as both were absent in Am80-treated IL-6 knock-out mice.ConclusionsThis study presents novel data showing a pro-inflammatory role of RAR ligands in T. muris infection, and implies an undesirable effect for the administration of vitamin A during chronic helminth infection.

Anti-inflammatory effects of infliximab in mice are independent of TNFα neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated ex vivo using enzyme‐linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.