Statins are well-known cholesterol lowering drugs targeting HMG-CoA-reductase, reducing the risk of coronary disorders and hypercholesterolemia. Statins are also involved in immunomodulation, which might influence the outcome of bacterial infection. Hence, a possible effect of statin treatment on Listeriosis was explored in mice. Statin treatment prior to subsequent L. monocytogenes infection strikingly reduced bacterial burden in liver and spleen (up to 100-fold) and reduced histopathological lesions. Statin-treatment in infected macrophages resulted in increased IL-12p40 and TNF-α and up to 4-fold reduced bacterial burden within 6 hours post infection, demonstrating a direct effect of statins on limiting bacterial growth in macrophages. Bacterial uptake was normal investigated in microbeads and GFP-expressing Listeria experiments by confocal microscopy. However, intracellular membrane-bound cholesterol level was decreased, as analyzed by cholesterol-dependent filipin staining and cellular lipid extraction. Mevalonate supplementation restored statin-inhibited cholesterol biosynthesis and reverted bacterial growth in Listeria monocytogenes but not in listeriolysin O (LLO)-deficient Listeria. Together, these results suggest that statin pretreatment increases protection against L. monocytogenes infection by reducing membrane cholesterol in macrophages and thereby preventing effectivity of the cholesterol-dependent LLO-mediated phagosomal escape of bacteria.
Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.
Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.
The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.
Nematode haemoglobins are fascinating molecules with unusually high affinity for oxygen. This is one example of many unique adaptations that nematodes have acquired to survive in their hosts, as nematode haemoglobin is thought to sequester oxygen to maintain an anaerobic environment, and can break down nitric oxide (NO) and hydrogen peroxide produced by host defences. This study describes the characterization of nematode haemoglobins using a novel monoclonal antibody (anti-Hb) generated against Anisakis haemoglobin, which was found to be highly expressed in stage 3 larvae and associated with the excretory-secretary ducts. Anisakis haemoglobin is an IgE-binding molecule in infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE in human sera. Finally, passive immunization of mice with anti-Hb provided protection against Nippostrongylus brasiliens (rodent hookworm), with mice showing reduced worm burden and enhanced Th2 responses, showing that haemoglobin may be a good vaccine target in some nematodes. The monoclonal antibody generated in this study will be useful in further studies to examine the biology of nematode haemoglobins.
Gut-dwelling helminthes induce potent IL-4 and IL-13 dominated type 2 T helper cell (TH2) immune responses, with IL-13 production being essential for Nippostrongylus brasiliensis expulsion. This TH2 response results in intestinal inflammation associated with local infiltration by T cells and macrophages. The resulting increased IL-4/IL-13 intestinal milieu drives goblet cell hyperplasia, alternative macrophage activation and smooth muscle cell hypercontraction. In this study we investigated how IL-4-promoted T cells contributed to the parasite induced effects in the intestine. This was achieved using pan T cell-specific IL-4 receptor alpha-deficient mice (iLckcreIL-4Rα−/lox) and IL-4Rα-responsive control mice. Global IL-4Rα−/− mice showed, as expected, impaired type 2 immunity to N. brasiliensis. Infected T cell-specific IL-4Rα-deficient mice showed comparable worm expulsion, goblet cell hyperplasia and IgE responses to control mice. However, impaired IL-4-promoted TH2 cells in T cell-specific IL-4Rα deficient mice led to strikingly reduced IL-4 production by mesenteric lymph node CD4+ T cells and reduced intestinal IL-4 and IL-13 levels, compared to control mice. This reduced IL-4/IL-13 response was associated with an impaired IL-4/IL-13-mediated smooth muscle cell hypercontractility, similar to that seen in global IL-4Rα−/− mice. These results demonstrate that IL-4-promoted T cell responses are not required for the resolution of a primary N. brasiliensis infection. However, they do contribute significantly to an important physiological manifestation of helminth infection; namely intestinal smooth muscle cell-driven hypercontractility.
Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.
Cutaneous leishmaniasis is endemic in tropical and subtropical regions of the world. Effective vaccination strategies are urgently needed because of the emergence of drug-resistant parasites and severe side effects of chemotherapy. We previously established a DC-based vaccination strategy to induce complete and long-lasting immunity to experimental leishmaniasis using Leishmania major antigen-loaded and CpG oligodeoxynucleotide-activated DC as a vaccine carrier. In the present study we investigated the role of IL-4Rα-mediated instruction of the vaccinating DC and the host DC during induction of protection against leishmaniasis. The results demonstrate that IL-4Rα signaling in DC used as vaccine carrier plays an important role in induction of protective immunity against L. major infection, as only mice vaccinated with IL-4 responder DC are able to trigger effective Th1-mediated immunity. The immunity is hallmarked by high levels of L. major-induced bioactive IL-12 production in the lymph nodes of vaccinated mice. Together, these findings suggest that IL-4 is a strong adjuvant to induce Th1-biased immunity against leishmaniasis and possibly other infections with intracellular pathogens, indicating that IL-4 needs to be considered in the development of efficient cell-mediated vaccination strategies.
All homeotherms utilize thermogenesis to maintain core body temperature, ensuring that cellular functions and physiologic processes can ensue in cold environments1-3. In the prevailing model, when the hypothalamus senses cold temperatures, it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue (BAT) and white adipose tissue (WAT)4,5. Acting via the β3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes6, whereas it stimulates the expression of thermogenic genes, such as PPARγ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1), and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes7-9. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report an unexpected requirement for the interleukin 4 (IL4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Cold exposure rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in BAT and lipolysis in WAT. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL4 increased thermogenic gene expression, fatty acid mobilization, and energy expenditure, all in a macrophage-dependent manner. We have thus discovered a surprising role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.
C-type lectin receptors (CLRs) that couple with the kinase Syk are major pattern recognition receptors for the activation of innate immunity and host defense. CLRs recognize fungi and other forms of microbial or sterile danger, and they induce inflammatory responses through the adaptor protein Card9. The mechanisms relaying CLR proximal signals to the core Card9 module are unknown. Here we demonstrated that protein kinase C-δ (PKCδ) was activated upon Dectin-1-Syk signaling, mediated phosphorylation of Card9 at Thr231, and was responsible for Card9-Bcl10 complex assembly and canonical NF-κB control. Prkcd−/− dendritic cells, but not those lacking PKCα, PKCβ, or PKCθ, were defective in innate responses to Dectin-1, Dectin-2, or Mincle stimulation. Moreover, Candida albicans-induced cytokine production was blocked in Prkcd−/− cells, and Prkcd−/− mice were highly susceptible to fungal infection. Thus, PKCδ is an essential link between Syk activation and Card9 signaling for CLR-mediated innate immunity and host protection.
► Dectin-1-Syk stimulation activates PKCδ in dendritic cells ► PKCδ controls Card9-Bcl10 complex assembly for canonical NF-κB activation ► Prkcd−/− cells are defective in Dectin-1-, Dectin-2-, or Mincle-triggered responses ► PKCδ is essential for innate antifungal immunity and host protection in vivo
Work in large part on Leishmania major in the 1980s identified two distinct apparently counter-regulatory CD4+ T cell populations, T helper (h)1 and Th2, that controlled resistance/susceptibility to infection respectively. However, the generation of IL-4−/− mice in the 1990s questioned the paramount role of this Th2 archetypal cytokine in the non-healing response to Leishmania infection. The more recent characterization of CD4+ T cell regulatory populations and further effector CD4+ T helper populations, Th17, Th9, and T follicular (f)h cells as well as the acknowledged plasticity in T helper cell function has further added to the complexity of host pathogen interactions. These interactions are complicated by the multiplicity of cells that respond to CD4+ T cell subset signatory cytokines, as well as the diversity of Leishmania species that are often subject to significantly different immune-regulatory controls. In this article we review current knowledge with regard to the role of CD4+ T cells and their products during Leishmania infection. In particular we update on our studies using conditional IL-4Rα gene-deficient mice that have allowed dissection of the cell interplay dictating the disease outcomes of the major Leishmania species infecting humans.
Leishmania; T helper 1; T helper 2; T regulatory cells; T helper 17; T follicular helper cells; interleukin-4; interferon-gamma
IL-4Rα expression on airway smooth muscle cells is sufficient for the development of airway hyperresponsiveness.
Production of the cytokines IL-4 and IL-13 is increased in both human asthma and mouse asthma models, and Stat6 activation by the common IL-4/IL-13R drives most mouse model pathophysiology, including airway hyperresponsiveness (AHR). However, the precise cellular mechanisms through which IL-4Rα induces AHR remain unclear. Overzealous bronchial smooth muscle constriction is thought to underlie AHR in human asthma, but the smooth muscle contribution to AHR has never been directly assessed. Furthermore, differences in mouse versus human airway anatomy and observations that selective IL-13 stimulation of Stat6 in airway epithelium induces murine AHR raise questions about the importance of direct IL-4R effects on smooth muscle in murine asthma models and the relevance of these models to human asthma. Using transgenic mice in which smooth muscle is the only cell type that expresses or fails to express IL-4Rα, we demonstrate that direct smooth muscle activation by IL-4, IL-13, or allergen is sufficient but not necessary to induce AHR. Five genes known to promote smooth muscle migration, proliferation, and contractility are activated by IL-13 in smooth muscle in vivo. These observations demonstrate that IL-4Rα promotes AHR through multiple mechanisms and provide a model for testing smooth muscle–directed asthma therapeutics.
Airflow in the lungs of patients with allergic asthma is impaired by excessive mucus production and airway smooth muscle contractions. Elevated levels of the cytokines IL-4 and IL-13 are associated with this pathology. In vitro studies have suggested that IL-4 receptor alpha (IL-4Rα) signalling on smooth muscle cells is critical for airway inflammation and airway hyperresponsiveness.
In order to define the contribution of IL-4 and IL-13 to the onset of asthmatic pathology the role of their key receptor IL-4Rα in smooth muscle cells was examined in vivo.
By using transgenic SMC-MHCcreIL-4Rα−/lox mice deficient for IL-4Rα in smooth muscle cells, in vivo effects of impaired IL-4Rα signalling in smooth muscle cells on the outcome of asthmatic disease were investigated for the first time. Allergic asthma was introduced in mice by repeated sensitisation with ovalbumin/aluminium hydroxide on days 0, 7 and 14 followed by intranasal allergen challenge on days 21–23. Mice were investigated for the presence of airway hyperresponsiveness, airway inflammation, allergen specific antibody production, Th2 type cytokine responses and lung pathology.
Airway hyperresponsiveness, airway inflammation, mucus production, Th2 cytokine production and specific antibody responses were unaffected in SMC-MHCcreIL-4Rα−/lox mice when compared to control animals.
The impairment of IL-4Rα on smooth muscle cells had no effect on major aetiological markers of allergic asthma. These findings suggest that IL-4Rα responsiveness in airway smooth muscle cells during the early phase of allergic asthma is not, as suggested, necessary for the outcome of the disease.
Therapies targeting the IL-4Rα might have no direct effect on smooth muscle cells in an allergic asthma response.
Smooth muscle cell; Allergy; Asthma; Cytokine Receptors; IL-4; IL-13; gene-deficient mice
Previous studies comparing interleukin 4 receptor α (IL-4Rα)-/- and interleukin 4 (IL-4)-/- BALB/c mice have indicated that interleukin 13 (IL-13), whose receptor shares the IL-4Rα subunit with IL-4, plays a protective role during visceral leishmaniasis. We demonstrate that IL-13-/- BALB/c mice were less able to control hepatic growth of Leishmania donovani compared with wild-type mice. This correlated with significantly retarded granuloma maturation in IL-13-/- mice, defective interferon γ (IFN-γ) production, and elevated IL-4 and interleukin 10 (IL-10) levels. L. donovani–infected IL-13-/- mice also responded poorly to sodium stibogluconate-mediated chemotherapy compared with wild-type BALB/c mice. Because murine lymphocytes do not have IL-13 receptors, we examined the ability of macrophage/neutrophil-specific IL-4Rα-/- mice to control primary infection with L. donovani and to respond to chemotherapy. Macrophage/neutrophil-specific IL-4Rα-/- mice were as resistant to leishmaniasis as wild-type mice, and chemotherapy retained its efficacy. Consequently, in L. donovani infected BALB/c mice, IL-13 promotes hepatic granuloma formation and controls parasite burdens independently of direct effects on macrophages/neutrophils.
Infection of the mammalian host by schistosome larvae occurs via the skin, although
nothing is known about the development of immune responses to multiple exposures of
schistosome larvae, and/or their excretory/secretory (E/S) products. Here, we show
that multiple (4x) exposures, prior to the onset of egg laying by adult worms,
modulate the skin immune response and induce CD4+ cell
hypo-responsiveness in the draining lymph node, and even modulate the formation of
hepatic egg-induced granulomas. Compared to mice exposed to a single infection (1x),
dermal cells from multiply infected mice (4x), were less able to support lymph node
cell proliferation. Analysis of dermal cells showed that the most abundant in 4x mice
were eosinophils (F4/80+MHC-II−), but they did not
impact the ability of antigen presenting cells (APC) to support lymphocyte
proliferation to parasite antigen in vitro. However, two other cell
populations from the dermal site of infection appear to have a critical role. The
first comprises arginase-1+, Ym-1+ alternatively
activated macrophage-like cells, and the second are functionally compromised
MHC-IIhi cells. Through the administration of exogenous IL-12 to
multiply infected mice, we show that these suppressive myeloid cell phenotypes form
as a consequence of events in the skin, most notably an enrichment of IL-4 and IL-13,
likely resulting from an influx of RELMα-expressing eosinophils. We further
illustrate that the development of these suppressive dermal cells is dependent upon
IL-4Rα signalling. The development of immune hypo-responsiveness to schistosome
larvae and their effect on the subsequent response to the immunopathogenic egg is
important in appreciating how immune responses to helminth infections are modulated
by repeated exposure to the infective early stages of development.
Schistosomiasis is a major helminth disease that infects more than 200 million people
in the tropics. Free-swimming aquatic cercariae infect through the skin after contact
with contaminated water, and in endemic areas this can occur frequently. However,
nothing is known about how multiple exposures affects innate immunity in the skin,
and/or whether it impacts the acquired immune response. Consequently, we have
developed an infection model in the mouse to examine the immune response to multiple
infections prior to the production of eggs. We show that multiple exposures to
schistosome larvae cause lymphocyte hypo-responsiveness, partly mediated by
macrophages and dendritic cells from the skin which have a
‘down-regulated’ phenotype and are not able to act as efficient antigen
presenting cells (APCs). These regulated APCs are conditioned amongst high levels of
the cytokines IL-4 and IL-13 which follow an influx of abundant eosinophils. In the
absence of the regulatory APCs, and in the absence of the common receptor chain for
IL-4 and IL-13 (i.e. IL-4Rα), lymphocyte proliferation is
restored. These findings are important in understanding how dermal immune responses
are modulated so that we can devise new strategies for vaccine delivery, or the
treatment of chronic inflammatory conditions of the skin.
Infection with the fish parasite Anisakis following exposure to contaminated fish can lead to allergic reactions in humans. The present study examined the immunological mechanisms underlying the development of allergic airway inflammation in mice after different routes of sensitization to Anisakis. Wild-type and interleukin-4 receptor alpha (IL-4Rα)-deficient BALB/c mice were sensitized intraperitoneally with live or heat-killed Anisakis larvae or by intranasal administration of an Anisakis extract and were subsequently challenged intranasally with an Anisakis extract. Both routes of sensitization induced IL-4Rα-dependent allergic airway responses, whereas allergen-specific antibody responses developed only when mice were sensitized intraperitoneally. Intranasal sensitization induced airway hyperresponsiveness (AHR) in wild-type mice only, showing that AHR was IL-4/IL-13 dependent. Unexpectedly, infection with Anisakis larvae induced AHR in both wild-type and IL-4Rα-deficient mice. IL-4Rα-independent AHR was mediated by gamma interferon (IFN-γ), as evidenced by the fact that in vivo neutralization of IFN-γ abrogated AHR. Together, these results demonstrate that both infection with larvae and inhalational exposure to Anisakis proteins are potent routes of allergic sensitization to Anisakis, explaining food- and work-related allergies in humans. Importantly for diagnosis, allergic airway inflammation can be independent of detectable Anisakis-specific antibodies. Moreover, depending on the route of sensitization, AHR can be induced either by IL-4/IL-13 or by IFN-γ.
There is interest in identifying the pattern recognition receptors involved in initiating protective or non-protective host responses to Mycobacterium tuberculosis (Mtb). Here we explored the role of the Syk/CARD9-coupled receptor, Dectin-1, using an aerosol model of Mtb infection in wild-type and Dectin-1 deficient mice. We observed a reduction in pulmonary bacilli burdens in the Dectin-1 deficient animals, but this did not correlate with significant changes in pulmonary pathology, cytokine levels or ability of these animals to survive the infection. Thus Dectin-1 makes a minor contribution to susceptibility to Mtb infections in mice.
C-type lectin receptors; Syk; CARD9; Dectin-1; Pulmonary disease; Inflammation
Immunologically intact BALB/c mice infected with Leishmania mexicana develop non-healing progressively growing lesions associated with a biased Th2 response while similarly infected IL-4Rα-deficient mice fail to develop lesions and develop a robust Th1 response. In order to determine the functional target(s) for IL-4/IL-13 inducing non-healing disease, the course of L. mexicana infection was monitored in mice lacking IL-4Rα expression in specific cellular compartments. A deficiency of IL-4Rα expression on macrophages/neutrophils (in LysMcreIL-4Rα−/lox animals) had minimal effect on the outcome of L. mexicana infection compared with control (IL-4Rα−/flox) mice. In contrast, CD4+ T cell specific (LckcreIL-4Rα−/lox) IL-4Rα−/− mice infected with L. mexicana developed small lesions, which subsequently healed in female mice, but persisted in adult male mice. While a strong Th1 response was manifest in both male and female CD4+ T cell specific IL-4Rα−/− mice infected with L. mexicana, induction of IL-4 was manifest in males but not females, independently of CD4+ T cell IL-4 responsiveness. Similar results were obtained using pan-T cell specific (iLckcreIL-4Rα−/lox) IL-4Rα−/− mice. Collectively these data demonstrate that upon infection with L. mexicana, initial lesion growth in BALB/c mice is dependent on non-T cell population(s) responsive to IL-4/IL-13 while progressive infection is dependent on CD4+ T cells responsive to IL-4.
Leishmania species are parasites, transmitted by sandflies which are of extensive public health importance in the tropical and subtropical regions of the world. A large number of distinct Leishmania species cause cutaneous disease and the vast majority of studies utilize the caustive agent of Old World cutaneous leishmaniasis, L. major. Other species, for example, L. mexicana, are associated with quite different patterns of disease following infection of mice when compared with L. major. Thus, while susceptible BALB/c mice deficient in the ability to respond to the cytokines IL-4/IL-13 are not protected against development of cutaneous leishmaniasis caused by L. major they are totally resistant to infection with L. mexicana. Here we describe the outcome of L. mexicana infection in BALB/c mice with cell-specific deletion of the receptor for IL-4/IL-13 on macrophages/neutrophils or T helper cells. Infections develop in both mutants but lesion growth is controlled only in T cell specific knockouts and female but not male mice heal. Male but not female T cell specific knockouts maintain a strong IL-4/IL-13 response. This highlights the role of IL4/IL-13 in driving a non-healing response and may in part explain why human males are more susceptible to this infection than females.
Th2 cells drive protective immunity against most parasitic helminths, but few mechanisms have been demonstrated that facilitate pathogen clearance. We show that IL-4 and IL-13 protect against intestinal lumen-dwelling worms primarily by inducing intestinal epithelial cells (IECs) to differentiate into goblet cells that secrete resistin-like molecule (RELM) β. RELM-β is essential for normal spontaneous expulsion and IL-4–induced expulsion of Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which both live in the intestinal lumen, but it does not contribute to immunity against Trichinella spiralis, which lives within IEC. RELM-β is nontoxic for H. polygyrus in vitro but directly inhibits the ability of worms to feed on host tissues during infection. This decreases H. polygyrus adenosine triphosphate content and fecundity. Importantly, RELM-β–driven immunity does not require T or B cells, alternative macrophage activation, or increased gut permeability. Thus, we demonstrate a novel mechanism for host protection at the mucosal interface that explains how stimulation of epithelial cells by IL-4 and IL-13 contributes to protection against parasitic helminthes that dwell in the intestinal lumen.
IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.
Schistosomiasis is a tropical disease caused by one of the species of the parasitic worm Schistosoma which infects over 200 million people worldwide. Signalling via the IL-4 receptor alpha (IL-4Rα), which is the common receptor chain for the ligands IL-4 and IL-13, is essential for inducing protective Type 2 immune response and granuloma formation in response to the parasite eggs. In experimental Schistosoma mansoni infection and egg-induced inflammation studies with cell type-specific IL-4Rα deficient mice, the role of IL-4Rα-activated alternative macrophages (aaMφ) and IL-4Rα-responsive T cells was investigated with focus on the control of hepatic inflammation and granuloma formation. Interestingly, aaMφ were not essential for the cellular composition or the Th1/Th2 cytokine profile in liver granulomas. In contrast, IL-4Rα-dependent T cell responses were important for predominant Th2 and IL-10 responses, as well as the presence of aaMφ in the granulomas, avoiding major disruption in the granuloma cell composition. Moreover, a macrophage subpopulation was identified and those cells expressed the two aaMφ markers, mannose receptor- and Ym1 in an IL-4Rα-independent but IL-10-dependent manner. These cells might be involved in the control of inflammation.
The costimulatory receptor CD28 and IL-4Rα-containing cytokine receptors play key roles in controlling the size and quality of pathogen-specific immune responses. Thus, CD28-mediated costimulation is needed for effective primary T-cell expansion and for the generation and activation of regulatory T-cells (Treg cells), which protect from immunopathology. Similarly, IL-4Rα signals are required for alternative activation of macrophages, which counteract inflammation by type 1 responses. Furthermore, immune modulation by CD28 and IL-4 is interconnected through the promotion of IL-4 producing T-helper 2 cells by CD28 signals. Using conditionally IL-4Rα and CD28 deleting mice, as well as monoclonal antibodies, which block or stimulate CD28, or mAb that deplete Treg cells, we have studied the roles of CD28 and IL-4Rα in experimental mouse models of virus (influenza), intracellular bacteria (L. monocytogenes, M. tuberculosis), and parasite infections (T. congolense, L. major). We observed that in some, but not all settings, Treg cells and type 2 immune deviation, including activation of alternative macrophages can be manipulated to protect the host either from infection or from immunopathology with an overall beneficial outcome. Furthermore, we provide direct evidence that secondary CD8 T-cell responses to i.c. bacteria are dependent on CD28-mediated costimulation.
CD28; Costimulation; IL-4; IL-4R; Alternatively activated macrophages; Mouse models; Conditional knockout; Monoclonal antibodies; Regulatory T-cells; Influenza; L. monocytogenes; M. tuberculosis; T. congolense; L. major
The interaction between Mycobacterium tuberculosis (Mtb) and host cells is complex and far from being understood. The role of the different receptor(s) implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.
Methods and Findings
To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1) as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1−/− or wild type mice in vitro. Moreover, when wild type and SR-B1−/− animals were infected with a low dose of Mtb (100 CFU/mouse) there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNγ, and IL10 were observed in SR-B1−/− mice following infection with a high dose of Mtb (1000 CFU/mouse), which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.
SR-B1 is involved in mycobacterial recognition, but this receptor plays only a minor role in anti-mycobacterial immunity in vivo. Like many other receptors for these pathogens, the loss of SR-B1 can be functionally compensated for under normal conditions.
Genetic background influences the outcome of L. major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-γ-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-γ-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-γ-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the lymph nodes and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3+ T cells in regional lymph nodes and lesions whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naïve BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-γ and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major-susceptibility.
Chemokines; cell surface markers; T cells; Th1/Th2 cells
The role of CD4+ T-cell interleukin-4 (IL-4) receptor alpha (IL-4Rα) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4+ T-cell IL-4Rα knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4+ T-cell IL-4Rα KO mice, the associated lung pathology was reduced. Infected CD4+ T-cell IL-4Rα KO mice showed abrogation of airway mucus production. Furthermore, CD4+ T-cell IL-4Rα KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4+ T cells was reduced in infected CD4+ T-cell IL-4Rα KO mice. Restimulation of tissue-derived CD4+ T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4+ T-cell IL-4Rα KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4+ T-cell IL-4Rα KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4+ T cells depleted showed that CD4+ T cells were a key TH2 cytokine source. These data demonstrated that CD4+ T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.
Obesity and insulin resistance, cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation1,2. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance3,4. For instance, transgenic expression of Mcp1 in adipose tissue increases macrophage infiltration, inflammation, and insulin resistance5,6. Conversely, disruption of Mcp1 or its receptor, Ccr2, impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity5,7. These findings together suggest a correlation between adipose tissue macrophage content (ATM) and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment8. While Mcp-1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage5,8, resident macrophages, such as those present in adipose tissue of lean mice, display the alternatively activated phenotype9. Despite their higher reparative capacity10, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of peroxisome proliferator activated receptor-γ (PPARγ), we show here that PPARγ is required for maturation of alternatively activated macrophages. Disruption of PPARγ in myeloid cells impairs alternative macrophage activation, thereby predisposing these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings demonstrate that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.
African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (µMT) and IgM-deficient (IgM−/−) mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei–infected µMT and IgM−/− mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in µMT mice as well as in IgM−/− mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection.
African trypanosomiasis is a disease caused by different species of extracellular flagellated protozoan trypanosome parasites. Trypanosomes have developed a mechanism of regular antigenic variation of their variant-specific surface glycoprotein (VSG) coat which allows chronic infection. Replacement of this coat occurs at rapid regular time intervals, allowing the parasite to escape from an effective host antibody responses. So far, primary T-cell independent antibody responses have been described to constitute the main host defense mechanism, relying largely on IgM antibody induction. Using genetically engineered B lymphocyte- or IgM-deficient mouse strains, we show that lack of B-cells or IgM did not prevent infection-associated anemia. More importantly, we show that in the absence of IgM, parasitemia was controlled almost as well as in wild-type mice, with only slightly increased mortality. In addition, we show in vivo that antigenic variation is not affected by the lack of IgM.
Intestinal mucus production by hyperplasic goblet cells is a striking pathological feature of many parasitic helminth infections and is related to intestinal protection and worm expulsion. Induction of goblet cell hyperplasia is associated with TH2 immune responses, which in helminth infections are controlled primarily by IL-13, and also IL-4. In the study presented here we examine the goblet cell hyperplasic response to three experimental parasitic helminth infections; namely Nippostrongylus brasiliensis, Syphacia obvelata and Schistosoma mansoni.
As expected N. brasiliensis infection induced a strong goblet cell hyperplasia dependent on IL-4/IL-13/IL-4Rα expression. In contrast, and despite previously published transiently elevated IL-4/IL-13 levels, S. obvelata infections did not increase goblet cell hyperplasia in the colon. Furthermore, induction of goblet cell hyperplasia in response to S. mansoni eggs traversing the intestine was equivalent between BALB/c, IL-4/IL-13-/- and IL-4Rα-/- mice.
Together these data demonstrate that intestinal goblet cell hyperplasia can be independent of TH2 immune responses associated with parasitic helminth infections.
We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) γ1 or μ heavy chains. Progenitor cells expressing γ1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro– to pre–B cell transition. Accordingly, γ1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Igα cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca2+ response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca2+ response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the γ1 chain can exert a unique signaling function that can partially replace that of the Igα/β heterodimer in B cell maintenance and may contribute to memory B cell physiology.
Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17–producing T cells. We have generated IL-25–deficient (il25−/−) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25−/− mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17–, IFNγ-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNγ in il25−/− mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1β, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.