Effects of intestinal worms on skin immunity and control of co-infection

Abstract: Intestinal helminth infections remain a major health concern in developing areas of the world. Consequences of infection range from gastrointestinal discomfort to systemic manifestations. It has been suggested that individuals infected with intestinal helminths are more susceptible to other infections and mount weaker immunity to vaccination. Regions heavily burdened by intestinal helminths geographically coincide with areas plagued by infections with mycobacteria and the protozoan parasite Leishmania spp, causing tuberculosis (TB) and leishmaniasis, respectively. Further, it has been reported that people carrying intestinal worms mount weaker immune responses to the intradermally administered tuberculosis vaccine Mycobacterium bovis Bacillus Calmette Guérin (BCG). Intestinal helminth infections induce type 2 responses that aid in the expulsion of the worms, but also regulatory responses that facilitate chronicity of the worm infection. Both type 2 and regulatory responses are known to counteract type 1 immune responses required for protection against the intracellular pathogens mycobacteria and Leishmania, suggesting that worm infection may dampen protection to these infections. Yet, little is known about the systemic implications of intestinal worm infection, and the aim of the work in this thesis was to investigate the effects of intestinal helminth infection on skin immunity and control of co-infection. To this end, mice were infected with the strictly intestinal nematode Heligmosomoides polygyrus, and at various time points after infection subjected to secondary infection, immunization, or were culled. At the end of each experiment, composition and function of immune cells in skin, skin-draining lymph nodes (LNs), liver, spleen and other tissues involved in responses to secondary infection, were analysed. In Paper I, we found that mice infected with H. polygyrus were more susceptible to systemic infection with BCG and skin infection with Leishmania major. Increased susceptibility to BCG was accompanied by weaker IFN-γ production and fewer mycobacteria-specific transgenic p25 cells in spleen, and less inos expression and granuloma formation in livers. Delayed type hypersensitivity responses (DTH) induced in ears to BCG and L. major-derived antigens were dampened. Dendritic cell (DC) migration from footpad skin to the draining LN was reduced in worm-infected mice, as well as in mice where the footpad skin had been preconditioned with either H. polygyrus excretory-secretory (HES) products or recombinant human transforming growth factor β (TGF-β). In vitro, BCG-induced IFN-γ production by mycobacteria-specific T cells was reduced by HES, soluble worm antigens, or by TGF-β. This led us to hypothesize that H. polygurus-induced reduction of immunity to the T helper cell type 1(TH1)-controlled organisms mycobacterium BCG and L. major was mediated by enhanced TGF-β production in worm-infected mice. In Paper II, we saw that (similar to the situation with H. polygyrus – BCG co-infected animals) worm-infected mice were more susceptible to systemic infection with the TH1- controlled pathogen Leishmania donovani. Reduced protection was accompanied by lower inos levels and granuloma formation in livers and higher il10 levels in spleens. In Paper III, we sought for the explanation to the weaker skin immunity seen in worminfected mice (in Paper I). We found that mice infected with H. polygyrus had substantially smaller skin-draining LNs compared to worm-free animals. Both T cell and B cells were fewer, whereas no significant difference was observed in myeloid and stromal cell populations. As mentioned, numbers of DCs migrating from BCG-injected skin as well as p25 cells were less in skin-draining LNs of worm-infected mice. Notably however, numbers were directly proportional to the total number of cells in that particular LN. This led us to hypothesize that cells enter or are retained in an LN dependent on the original size of that node. As oppose to the atrophic skin draining LNs, the gut-draining mesenteric LNs were instead (as expected) dramatically increased in size. The lymphocyte pool cannot expand without limitation, and we suggested that worm-induced expansion of one LN occurred at the expense of other LN. Removal of worms restored the sizes of the non-draining nodes. However, this took time, since (according to out hypothesis) the atrophy of skin draining LNs and hyperplasia of mesenteric LN in itself decreased or increased infiltration or retention of cells into the respective nodes, maintaining this new “homeostasis”. In the last paper, Paper IV, we proceeded by investigating immune cells in the skin itself after H. polygyrus infection. We found that mice infected with H. polygyrus had fewer CD4+ cells producing IFN-γ in ear skin injected with whole cell lysate (WCL) from Mycobacterium tuberculosis in response to mycobacteria-specific ex-vivo re-stimulation, compared to wormfree mice. IFN-γ production was also lower in the contralateral, untouched ear. Interestingly however, the total number of CD4+ cells were higher in ear skin of worm-infected mice. CD4+ T cell numbers were also higher when comparing H. polygyrus-infected and noninfected animals without any skin stimulation, indicating that the intestinal infection, in itself, caused accumulation of CD4+ T cells in the skin. We found that the accumulated CD4+ T cells responded to H. polygyrus antigen by producing TH2 associated cytokines and that they remained in the skin for several weeks after removal of worms from the intestine. In accordance, skin-homing chemokine receptors were up-regulated on CD4+ T cells in the mesenteric LNs and blood. We hypothesized that the increased number of TH2 cells in the skin, in concert with the atrophy of skin draining LNs, were responsible for the lower protection to TH1-controlled organisms in the skin. In conclusion, mice chronically infected with the strictly intestinal nematode H. polygyrus were more susceptible to systemic and skin infection by TH1-controlled organisms compared to worm-free mice. We suggest that less inos and granuloma formation contributed to lower protection to systemic infection and that a combination of atrophic skin-draining LNs and increased numbers of TH2 cells in the skin caused weaker skin immunity. Taken together, this indicates that deworming may increase protection against secondary infection and increase beneficial effects of BCG vaccination.