Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths

Helminth infection is frequently associated with the expansion of regulatory T cells (Tregs) and suppression of immune responses to bystander antigens. We show that infection of mice with the chronic gastrointestinal helminth Heligmosomoides polygyrus drives rapid polyclonal expansion of Foxp3(+)Helios(+)CD4(+) thymic (t)Tregs in the lamina propria and mesenteric lymph nodes while Foxp3(+)Helios(-)CD4(+) peripheral (p)Treg expand more slowly. Notably, in partially resistant BALB/c mice parasite survival positively correlates with Foxp3(+)Helios(+)CD4(+) tTreg numbers. Boosting of Foxp3(+)Helios(+)CD4(+) tTreg populations by administration of recombinant interleukin-2 (rIL-2):anti-IL-2 (IL-2C) complex increased worm persistence by diminishing type-2 responsiveness in vivo, including suppression of alternatively activated macrophage and granulomatous responses at the sites of infection. IL-2C also increased innate lymphoid cell (ILC) numbers, indicating that Treg functions dominate over ILC effects in this setting. Surprisingly, complete removal of Tregs in transgenic Foxp3-DTR mice also resulted in increased worm burdens, with "immunological chaos" evident in high levels of the pro-inflammatory cytokines IL-6 and interferon-γ. In contrast, worm clearance could be induced by anti-CD25 antibody-mediated partial depletion of early Treg, alongside increased T helper type 2 responses and without incurring pathology. These findings highlight the overarching importance of the early Treg response to infection and the non-linear association between inflammation and the prevailing Treg frequency

A structurally distinct TGF-β mimic from an intestinal helminth parasite potently induces regulatory T cells.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-β signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-β or other members of the TGF-β family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host

Natural and Vaccine-Mediated Immunity to Salmonella Typhimurium is Impaired by the Helminth Nippostrongylus brasiliensis

The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization

Depletion efficacy of Treg in BALB/c DEREG and C57BL/6 DEREG mice.

<p>BALB/c (white bars), BALB/c DEREG (black bars), C57BL/6 (light grey bars), and C57BL/6 DEREG (dark grey bars) mice were treated with DT and infected s.c. with 2000 <i>S. ratti</i> iL3. Mice were sacrificed days 2 and 6 p.i. and peripheral blood lymphocytes, mesenteric lymph node cells and spleen cells were stained for Foxp3 and CD4. <b>A:</b> Experimental setup is shown. <b>B:</b> Shown are representative dotplots of CD4/Foxp3 staining on day 2 p.i. (upper panel) and day 6 p.i. (lower panel) in peripheral blood lymphocytes. <b>C–E:</b> Percentage of Foxp3⁺ cells in CD4⁺ T cells at indicated time points in PBL (<b>C</b>) mesenteric lymph nodes (<b>D</b>) and spleen (<b>E</b>). The experiment shown (n = 4) is representative for 3 repeats; Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, *** p≤0.001).</p

Depletion of IL-9 during <i>S. ratti</i> infection in BALB/c DEREG mice at different time points.

<p><b>A:</b> Experimental setup for α-IL-9 treatment either early (blue arrows) or late (grey arrows) in <i>S. ratti</i> infection is shown. <b>B:</b> Graph shows number of parasitic adults in the small intestine of BALB/c (white), Treg-depleted, isotype treated BALB/c DEREG (black), Treg-depleted early α-IL-9 treated BALB/c DEREG (dark blue) and Treg-depleted late α-IL-9 treated BALB/c DEREG (dark grey) mice on day 6 p.i. Shown are the combined results of three independent experiments (n = 14). <b>C:</b> Concentrations of MMCP-1 in the serum of infected mice on day 4 and day 6 p.i. are shown as combined results of two independent experiments (n = 9). Asterisks indicate significant difference of the mean analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, *** p≤0.001).</p

IL-9 administration or depletion during <i>S. ratti</i> infection.

<p><b>A:</b> Experimental setup for IL-9 treatment is shown. <b>B–C:</b> Number of parasitic adults in the small intestine of infected non-treated BALB/c (<b>B</b>, white) and C57BL/6 (<b>C</b>, light grey) mice or infected and IL-9 treated BALB/c (<b>B</b>, black) and C57BL/6 (<b>C</b>, dark grey) mice on day 6 p.i. Shown are the combined results of three independent experiments (n = 12). <b>D:</b> Experimental setup for α-IL-9 treatment is shown. <b>E–F:</b> Number of parasitic adults in the small intestine of infected isotype treated BALB/c (<b>D</b>, white) and C57BL/6 (<b>E</b>, light grey) mice or infected and α-IL-9 treated BALB/c (<b>D</b>, black) and C57BL/6 (<b>E</b>, dark grey) mice on day 6 p.i. <b>G–H</b>: Concentration of MMCP-1 in the serum of infected isotype treated or α-IL-9 treated BALB/c (<b>G</b>) and C57BL/6 (<b>H</b>) mice on day 4 and day 6 p.i. Shown are the combined results of two independent experiments (n = 8). Asterisks indicate significant differences of the mean analyzed by students <i>t</i> test (** p≤0.01, *** p≤0.001).</p

Treg expansion during <i>S. ratti</i> infection.

<p>BALB/c DEREG and C57BL/6 DEREG mice were infected s.c. with 2000 <i>S. ratti</i> iL3. Mice were sacrificed at the indicated time points. Lymph node cells were stained for CD4 and CD103 (<b>AB</b>) or CD4, Foxp3, Helios and Neuropilin (<b>C</b>). Gating strategy is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003913#ppat.1003913.s001" target="_blank">Figure S1</a>. <b>AB:</b> Graphs show numbers of Treg (upper panel, CD4⁺GFP⁺), Teff (middle panel, CD4⁺GFP⁻) and activated Treg (lower panel CD103⁺CD4⁺GFP⁺) in popliteal (<b>A</b>) and mesenteric (<b>B</b>) lymph nodes at indicated time points of infection in BALB/c DEREG (left) and C57BL/6 DEREG (right) mice. <b>C:</b> Graphs show frequency of Helios⁺ and Neuropilin⁺ cells in the CD4⁺Foxp3⁺ Treg population in mesenteric lymph nodes of BALB/c and C57BL/6 mice at the indicated time points of infection. Shown are the combined results of two independent experiments (<b>AB:</b> n = 7 and <b>C:</b> n = 4–6). Asterisks indicate significant difference of the mean of infected mice compared to naïve mice (day 0 p.i.) analyzed by one-way ANOVA with Bonferroni post test (* p≤0.05, ** p≤0.01, *** p≤0.001).</p