Elucidating different pattern of immunoregulation in BALB/c and C57BL/6 mice and their F1 progeny

© The Author(s) 2021. Open Access. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .Helminths are large multicellular parasites that infect one quarter of the human population. To prolong their survival, helminths suppress the immune responses of their hosts. Strongyloides ratti delays its expulsion from the gut by induction of regulatory circuits in a mouse strain-specific manner: depletion of Foxp3+ regulatory T cells (Treg) improves the anti-S. ratti immunity in BALB/c but not in C57BL/6 mice. In the current study we compare the hierarchy of immunoregulatory pathways in BALB/c, C57BL/6 mice and their F1 progeny (BALB/c × C57BL/6). Using multicolor flow cytometry, we show that S. ratti induces a distinct pattern of inhibitory checkpoint receptors by Foxp3+ Treg and Foxp3- T cells. Intensity of expression was highest in C57BL/6 and lowest in BALB/c mice, while the F1 cross had an intermediate phenotype or resembled BALB/c mice. Treg subsets expanded during infection in all three mouse strains. Similar to BALB/c mice, depletion of Treg reduced intestinal parasite burden and increased mucosal mast cell activation in S. ratti-infected F1 mice. Our data indicate that Treg dominate the regulation of immune responses in BALB/c and F1 mice, while multiple regulatory layers exist in C57BL/6 mice that may compensate for the absence of Treg.Open Access funding enabled and organized by Projekt DEAL.info:eu-repo/semantics/publishedVersio

IL-33 facilitates rapid expulsion of the parasitic nematode <i>Strongyloides ratti</i> from the intestine via ILC2- and IL-9-driven mast cell activation

Parasitic helminths are sensed by the immune system via tissue-derived alarmins that promote the initiation of the appropriate type 2 immune responses. Here we establish the nuclear alarmin cytokine IL-33 as a non-redundant trigger of specifically IL-9-driven and mast cell-mediated immunity to the intestinal parasite Strongyloides ratti. Blockade of endogenous IL-33 using a helminth-derived IL-33 inhibitor elevated intestinal parasite burdens in the context of reduced mast cell activation while stabilization of endogenous IL-33 or application of recombinant IL-33 reciprocally reduced intestinal parasite burdens and increased mast cell activation. Using gene-deficient mice, we show that application of IL-33 triggered rapid mast cell-mediated expulsion of parasites directly in the intestine, independent of the adaptive immune system, basophils, eosinophils or Gr-1+ cells but dependent on functional IL-9 receptor and innate lymphoid cells (ILC). Thereby we connect the described axis of IL-33-mediated ILC2 expansion to the rapid initiation of IL-9-mediated and mast cell-driven intestinal anti-helminth immunity

Retinoic acid drives intestine-specific adaptation of effector ILC2s originating from distant sites.

Adaptation of immune cells to tissue-specific microenvironments is a crucial process in homeostasis and inflammation. Here, we show that murine effector type 2 innate lymphoid cells (ILC2s) from various organs are equally effective in repopulating ILC2 niches in other anatomical locations where they adapt tissue-specific phenotypes of target organs. Single-cell transcriptomics of ILC2 populations revealed upregulation of retinoic acid (RA) signaling in ILC2s during adaptation to the small intestinal microenvironment, and RA signaling mediated reprogramming of kidney effector ILC2s toward the small intestinal phenotype in vitro and in vivo. Inhibition of intestinal ILC2 adaptation by blocking RA signaling impaired worm expulsion during Strongyloides ratti infection, indicating functional importance of ILC2 tissue imprinting. In conclusion, this study highlights that effector ILC2s retain the ability to adapt to changing tissue-specific microenvironments, enabling them to exert tissue-specific functions, such as promoting control of intestinal helminth infections

CD83 Modulates B Cell Function In Vitro: Increased IL-10 and Reduced Ig Secretion by CD83Tg B Cells

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell responses. Recently, we reported that CD83 also has a function on B cells: Ubiquitous transgenic (Tg) expression of CD83 interfered with the immunoglobulin (Ig) response to infectious agents and to T cell dependent as well as T cell independent model antigen immunization. Here we compare the function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells that display a drastically reduced CD83 expression. Correlating with CD83 expression, the basic as well as the lipopolysaccharide (LPS) induced expression of the activation markers CD86 and MHC-II are significantly increased in CD83Tg B cells and reciprocally decreased in CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR or TLR engagement by de novo protein synthesis. The forced premature overexpression of CD83 on the CD83Tg B cells results in reduced calcium signaling, reduced Ig secretion and a reciprocally increased IL-10 production upon in vitro activation. This altered phenotype is mediated by CD83 expressed on the B cells themselves, since it is observed in the absence of accessory cells. In line with this finding, purified CD83mu B cells displayed a reduced IL-10 production and slightly increased Ig secretion upon LPS stimulation in vitro. Taken together, our data strongly suggest that CD83 is expressed by B cells upon activation and contributes to the regulation of B cell function

<i>Strongyloides</i> questions-a research agenda for the future.

The Strongyloides genus of parasitic nematodes have a fascinating life cycle and biology, but are also important pathogens of people and a World Health Organization-defined neglected tropical disease. Here, a community of Strongyloides researchers have posed thirteen major questions about Strongyloides biology and infection that sets a Strongyloides research agenda for the future. This article is part of the Theo Murphy meeting issue 'Strongyloides: omics to worm-free populations'

Basophils are dispensable for the establishment of protective adaptive immunity against primary and challenge infection with the intestinal helminth parasite Strongyloides ratti.

Infections with helminth parasites are controlled by a concerted action of innate and adaptive effector cells in the frame of a type 2 immune response. Basophils are innate effector cells that may also contribute to the initiation and amplification of adaptive immune responses. Here, we use constitutively basophil-deficient Mcpt8-Cre mice to analyze the impact of basophils during initiation and execution of the protective type 2 responses to both, a primary infection and a challenge infection of immune mice with the helminth parasite Strongyloides ratti. Basophil numbers expanded during parasite infection in blood and mesenteric lymph nodes. Basophil deficiency significantly elevated intestinal parasite numbers and fecal release of eggs and larvae during a primary infection. However, basophils were neither required for the initiation of a S. ratti-specific cellular and humoral type 2 immune response nor for the efficient protection against a challenge infection. Production of Th2 cytokines, IgG1 and IgE as well as mast cell activation were not reduced in basophil-deficient Mcpt8-Cre mice compared to basophil-competent Mcpt8-WT littermates. In addition, a challenge infection of immune basophil-deficient and WT mice resulted in a comparable reduction of tissue migrating larvae, parasites in the intestine and fecal release of eggs and L1 compared to mice infected for the first time. We have shown previously that S. ratti infection induced expansion of Foxp3+ regulatory T cells that interfered with efficient parasite expulsion. Here we show that depletion of regulatory T cells reduced intestinal parasite burden also in absence of basophils. Thus basophils were not targeted specifically by S. ratti-mediated immune evasive mechanisms. Our collective data rather suggests that basophils are non-redundant innate effector cells during murine Strongyloides infections that contribute to the early control of intestinal parasite burden

Suppressed IgG response to TD vaccination 16 weeks after termination of <i>L. sigmodontis</i> infection.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (closed squares and circles) or left non-infected (white squares). Mice were allowed to naturally clear the infection. Microfilaraemia was monitored and the time point no MF was detectable in the blood of any previously infected mouse was defined as termination of infection. 16 weeks later non-infected mice (open squares, n = 14) and mice, which had terminated the infection (orange squares, n = 15) were vaccinated with 100 µg DNP-KLH/Alum i.p. An additional control group was allowed to clear the infection, but was not DNP-KLH/Alum-vaccinated (closed circles, n = 2). <b>(B)</b> Microfilaraemia of mice naturally infected with <i>L. sigmodontis</i> of one experiment representative for three independent repeats. <b>(C)</b> DNP-specific IgG1 and IgG2b was quantified in sera. Results are expressed as mean ± SEM of pooled data derived from three independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice (Two-way ANOVA).</p

Infection with <i>L. sigmodontis</i> suppresses humoral response to TD vaccination.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> (closed squares) or left non-infected (open squares; n = 10) and were vaccinated with 100 µg DNP-KLH/Alum i.p. at <b>(B)</b> day 0 (grey, n = 8–10), <b>(C)</b>) day 14 (black, n = 10), <b>(D)</b> day 30 (red, n = 10) or <b>(E)</b> day 60 (blue, n = 10) p.i. An additional control group was infected but not vaccinated (closed circles, n = 2–4). DNP-specific IgG1, IgG2a and IgG2b was detected in sera. Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice after vaccination with DNP-KLH (Two-way ANOVA).</p

Reduced numbers of vaccine-specific B cells in <i>L. sigmodontis</i>-infected mice.

<p>Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> or left non-infected and vaccinated with 30 µg DNP-KLH/Alum s.c. at day 60 p.i. PopLN cells from DNP-KLH-vaccinated non-infected (white bars) and <i>L. sigmodontis</i>-infected (black bars) mice were stained for CD19, PNA-binding, DNP-binding, IgM and IgG at day 14 post vaccination. Mice had been infected for 60 days at the time point of vaccination. <b>(A)</b> Gating strategy of CD19⁺ B lymphocytes. <b>(B)</b>Total cell numbers of DNP⁺PNA⁺ lymph node cells within the CD19⁺ (total), within the IgG⁺IgM⁺CD19⁺ and within the IgG⁺CD19⁺ gate (n = 10–14). Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences between non-infected and infected mice (student's t-test).</p

Spatial separation of nematode- and vaccine-specific responses.

<p><b>(A)</b> Diagram of the experimental setup. Six- to eight-week-old BALB/c mice were naturally infected with <i>L. sigmodontis</i> or left non-infected and vaccinated with 30 µg DNP-KLH/Alum s.c. at day 60 p.i. <b>(B)</b> Diagram of spatial separation of immune responses. <b>(C)</b> Splenocytes and draining popLN cells were cultured for 21 days at 37°C. Supernatants were analysed for DNP-specific and <i>L. sigmodontis</i>-specific IgG1, IgG2a and IgG2b. <b>(D)</b> Quantification of DNP-specific IgG1 and IgG2b in sera of <i>L. sigmodontis</i>-infected (blue squares, n = 14) and non-infected (white squares, n = 10) vaccinated mice. Control mice were infected but not vaccinated (black circles, n = 2). Results are expressed as mean ± SEM of pooled data derived from two independent experiments. Asterisks indicate significant differences of the mean of DNP-specific Ig in non-infected and infected mice (Two-way ANOVA).</p