Marc P. Hübner

Inhibition of type 1 diabetes in filaria-infected non-obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells

We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non‐obese diabetic (NOD) mice. Six‐week‐old NOD mice were sham‐infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis‐infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis‐infected mice had greater numbers of total islets and non‐infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin‐4 (IL‐4) and IL‐5 release from α‐CD3/α‐CD28‐stimulated splenocytes was greater in L. sigmodontis‐infected mice than in uninfected mice. Increased circulating levels of insulin‐specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis‐infected NOD mice was associated with significantly increased numbers of splenic CD4+ CD25+ FoxP3+ regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4+ CD25+ FoxP3+ regulatory T cells.

Helminth Protection against Autoimmune Diabetes in Nonobese Diabetic Mice Is Independent of a Type 2 Immune Shift and Requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4–deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4–competent NOD mice, IL-4–deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4–deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4–deficient NOD mice were accompanied by increases in CD4+CD25+Foxp3+ regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4+Foxp3+ cells. However, depletion of CD25+ cells in NOD mice or Foxp3+ T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.

Basophils Amplify Type 2 Immune Responses, but Do Not Serve a Protective Role, during Chronic Infection of Mice with the Filarial Nematode Litomosoides sigmodontis

Chronic helminth infections induce a type 2 immune response characterized by eosinophilia, high levels of IgE, and increased T cell production of type 2 cytokines. Because basophils have been shown to be substantial contributors of IL-4 in helminth infections, and because basophils are capable of inducing Th2 differentiation of CD4+ T cells and IgE isotype switching in B cells, we hypothesized that basophils function to amplify type 2 immune responses in chronic helminth infection. To test this, we evaluated basophil function using the Litomosoides sigmodontis filaria model of chronic helminth infection in BALB/c mice. Time-course studies showed that eosinophilia, parasite Ag-specific CD4+ T cell production of IL-4 and IL-5 and basophil activation and IL-4 production in response to parasite Ag all peak late (6–8 wk) in the course of L. sigmodontis infection, after parasite-specific IgE has become detectable. Mixed-gender and single-sex worm implantation experiments demonstrated that the relatively late peak of these responses was not dependent on the appearance of circulating microfilariae, but may be due to initial low levels of parasite Ag load and/or habitation of the developing worms in the pleural space. Depletion of basophils throughout the course of L. sigmodontis infection caused significant decreases in total and parasite-specific IgE, eosinophilia, and parasite Ag-driven CD4+ T cell proliferation and IL-4 production, but did not alter total worm numbers. These results demonstrate that basophils amplify type 2 immune responses, but do not serve a protective role, in chronic infection of mice with the filarial nematode L. sigmodontis.

CD200R surface expression as a marker of murine basophil activation

Background Basophils are increasingly recognized as playing important roles in the immune responses of allergic diseases and helminth infections. One of the main obstacles to studying basophils has been the lack of a simple and rapid assay to measure basophil activation in mice.

Chronic Helminth Infection Reduces Basophil Responsiveness in an IL-10–Dependent Manner

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.

Immune system development during early childhood in tropical Latin America: Evidence for the age-dependent down regulation of the innate immune response

The immune response that develops in early childhood underlies the development of inflammatory diseases such as asthma and there are few data from tropical Latin America (LA). This study investigated the effects of age on the development of immunity during the first 5 years of life by comparing innate and adaptive immune responses in Ecuadorian children aged 6–9 months, 22–26 months, and 48–60 months. Percentages of naïve CD4+ T cells declined with age while those of memory CD4+ and CD8+ T cells increased indicating active development of the immune system throughout the first five years. Young infants had greater innate immune responses to TLR agonists compared to older children while regulatory responses including SEB-induced IL-10 and percentages of FoxP3+ T-regulatory cells decreased with age. Enhanced innate immunity in early life may be important for host defense against pathogens but may increase the risk of immunopathology.

Chronic helminth infection does not exacerbate Mycobacterium tuberculosis infection.

Background Chronic helminth infections induce a Th2 immune shift and establish an immunoregulatory milieu. As both of these responses can suppress Th1 immunity, which is necessary for control of Mycobacterium tuberculosis (MTB) infection, we hypothesized that chronic helminth infections may exacerbate the course of MTB. Methodology/Principal Findings Co-infection studies were conducted in cotton rats as they are the natural host for the filarial nematode Litomosoides sigmodontis and are an excellent model for human MTB. Immunogical responses, histological studies, and quantitative mycobacterial cultures were assessed two months after MTB challenge in cotton rats with and without chronic L. sigmodontis infection. Spleen cell proliferation and interferon gamma production in response to purified protein derivative were similar between co-infected and MTB-only infected animals. In contrast to our hypothesis, MTB loads and occurrence and size of lung granulomas were not increased in co-infected animals. Conclusions/Significance These findings suggest that chronic filaria infections do not exacerbate MTB infection in the cotton rat model. While these results suggest that filaria eradication programs may not facilitate MTB control, they indicate that it may be possible to develop worm-derived therapies for autoimmune diseases that do not substantially increase the risk for infections.

Anti-FcεR1 antibody injections activate basophils and mast cells and delay Type 1 diabetes onset in NOD mice

Mounting evidence suggests that helminth infections protect against autoimmune diseases. As helminths cause chronic IgE-mediated activation of basophils and mast cells we hypothesized that continuous activation of these cells could prevent diabetes onset in nonobese diabetic (NOD) mice in the absence of infection. Anti-FcεR1 activated basophils and mast cells and resulted in the release of IL-4 and histamine into the bloodstream. Anti-FcεR1-treated NOD mice showed a type 2 shift in insulin-specific antibody production and exhibited significant delays in diabetes onset. IL-4 responses played a partial role as the protective effect of anti-FcεR1 therapy was diminished in IL-4-deficient NOD mice. In contrast, histamine signaling was not required as anti-FcεR1-mediated protection was not reduced in mice treated with histamine receptor blockers. These results demonstrate that anti-FcεR1 therapy delays diabetes onset in NOD mice and suggest that chronic basophil and mast cell activation may represent a new avenue of therapy for Th1-associated autoimmune diseases.

Litomosoides sigmodontis: a simple method to infect mice with L3 larvae obtained from the pleural space of recently infected jirds (Meriones unguiculatus).

Litomosoides sigmodontis is a filarial nematode that is used as a mouse model for human filarial infections. The life cycle of L. sigmodontis comprises rodents as definitive hosts and tropical rat mites as alternate hosts. Here, we describe a method of infecting mice with third stage larvae (L3) extracted from the pleural space of recently infected jirds (Meriones unguiculatus). This method enables infection of mice with a known number of L3 larvae without the time-consuming dissection of L3 larvae from mites and results in higher worm recovery and patency rates than conventional methods. Additionally, this method allows for geographical separation of the facility maintaining the L. sigmodontis life cycle from the institution at which mice are infected.

Chronic Filarial Infection Provides Protection against Bacterial Sepsis by Functionally Reprogramming Macrophages

Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) and the outcome of acute systemic inflammation caused by i.p. Escherichia coli injection was determined. L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in L.s.-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as L.s., signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from L.s.-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with L.s. extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a Wolbachia- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from L.s.-infected wild type, but not TLR2-deficient animals. In association, L.s. infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated E. coli-induced hypothermia and sepsis survival. These results indicate that chronic L.s. infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.