Innate immunity is regulated by cholinergic signalling through nicotinic acetylcholine receptors. We show here that signalling through the M3 muscarinic acetylcholine receptor (M3R) plays an important role in adaptive immunity to both Nippostrongylus brasiliensis and Salmonella enterica serovar Typhimurium, as M3R-/- mice were impaired in their ability to resolve infection with either pathogen. CD4 T cell activation and cytokine production were reduced in M3R-/- mice. Immunity to secondary infection with N. brasiliensis was severely impaired, with reduced cytokine responses in M3R-/- mice accompanied by lower numbers of mucus-producing goblet cells and alternatively activated macrophages in the lungs. Ex vivo lymphocyte stimulation of cells from intact BALB/c mice infected with N. brasiliensis and S. typhimurium with muscarinic agonists resulted in enhanced production of IL-13 and IFN-γ respectively, which was blocked by an M3R-selective antagonist. Our data therefore indicate that cholinergic signalling via the M3R is essential for optimal Th1 and Th2 adaptive immunity to infection.
Recent data indicate that acetylcholine (ACh), a neurotransmitter which regulates a variety of physiological functions, also influences the immune system, and that lymphocytes have the capacity to synthesise and release ACh, controlling local innate immune responses and suppressing inflammation. Thus far however there has been little evidence to suggest that ACh influences adaptive immunity, characterised by activation and effector functions of lymphocytes. We show here that during the immune response to two different pathogens, ACh signals through muscarinic receptors, and the M3 receptor subtype specifically, resulting in enhanced activation and cytokine production by ‘helper’ T lymphocytes which protect the host against infection.
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.
Vaccination studies in animal models have focused on understanding responses in young, previously naïve mice. In reality, humans are vaccinated or respond to infection in the context of a life-time of accumulated exposure to multiple, systemic infections and other vaccines, some of which are themselves attenuated live organisms. This is even more pronounced in areas that are endemic for infectious diseases. We wished to examine the impact infectious history can have on the immune response against infection and the efficacy of vaccination. To do this, we used two classes of pathogens that model clinically important invasive infections. One pathogen is the bacterium, Salmonella Typhimurium against which we have also developed an experimental porin vaccine, and the second is an invasive helminth, Nippostrongylus brasiliensis, that models aspects of hookworm infections. Our studies indicate that exposure to a second, unrelated pathogen can reduce the efficiency of immunity generated during natural infection and immunity generated after vaccination. These results are important as they help to identify potential strategies for improving immune-mediated control of infection and the success of vaccination in infection-endemic regions.
IL-4 and CSF-1 both contribute to macrophage proliferation during nematode infection, but IL-4 permits increased tissue macrophage density without the coincident monocyte infiltration associated with elevated CSF-1 levels.
Macrophages (MΦs) colonize tissues during inflammation in two distinct ways: recruitment of monocyte precursors and proliferation of resident cells. We recently revealed a major role for IL-4 in the proliferative expansion of resident MΦs during a Th2-biased tissue nematode infection. We now show that proliferation of MΦs during intestinal as well as tissue nematode infection is restricted to sites of IL-4 production and requires MΦ-intrinsic IL-4R signaling. However, both IL-4Rα–dependent and –independent mechanisms contributed to MΦ proliferation during nematode infections. IL-4R–independent proliferation was controlled by a rise in local CSF-1 levels, but IL-4Rα expression conferred a competitive advantage with higher and more sustained proliferation and increased accumulation of IL-4Rα+ compared with IL-4Rα− cells. Mechanistically, this occurred by conversion of IL-4Rα+ MΦs from a CSF-1–dependent to –independent program of proliferation. Thus, IL-4 increases the relative density of tissue MΦs by overcoming the constraints mediated by the availability of CSF-1. Finally, although both elevated CSF1R and IL-4Rα signaling triggered proliferation above homeostatic levels, only CSF-1 led to the recruitment of monocytes and neutrophils. Thus, the IL-4 pathway of proliferation may have developed as an alternative to CSF-1 to increase resident MΦ numbers without coincident monocyte recruitment.
IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28−/loxCre+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28−/loxCre+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5+ TFH cell population. Furthermore, total number of CD4+ T cells and B220+ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28−/− mice and tamoxifen treated CD28−/loxCre+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4+ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28−/loxCre+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.
CD28 is an important costimulatory molecule, involved in the activation of naive T cells, enhancing cytokine production, preventing T cell anergy and apoptosis. Furthermore, CD28 plays a crucial role in the organisation of secondary lymphoid tissue by assisting in the recruitment of T cells into the B cell follicles, thus promoting germinal center formation, isotype switching and B cell maturation. The requirement of CD28 costimulatory signalling during recall of memory responses against infections has remained controversial. Hence, here we utilised a mouse model that allowed for inducible deletion of the cd28 gene (CD28−/loxCre+/−) by oral administration of tamoxifen to resolve this controversy. CD28−/− mice and mice given tamoxifen prior to secondary infection failed to expel adult N. brasiliensis worms. This was related to reduced production of the Th2 cytokines IL-13 and IL-4, diminished type 2 antibody titres, and a reduced number of memory CD4+ T cells. In summary, CD28 is crucial for protection against N. brasiliensis secondary infection and plays a key role in the recruitment of TFH cells, memory CD4+ T cells and follicular B cells.
Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.
The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the α chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus over-production. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell–deficient mice by an IL-4 receptor α chain–dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.
The general paradigm is that monocytes are recruited to sites of inflammation and terminally-differentiate into macrophages. There has been no demonstration of proliferation of peripherally-derived inflammatory macrophages under physiological conditions. Here we show that proliferation of both bone marrow-derived inflammatory and tissue resident macrophage lineage branches is a key feature of the inflammatory process with major implications for the mechanisms underlying recovery from inflammation. Both macrophage lineage branches are dependent on M-CSF during inflammation, and thus the potential for therapeutic interventions is marked. Furthermore, these observations are independent of Th2 immunity. These studies indicate that the proliferation of distinct macrophage populations provides a general mechanism for macrophage expansion at key stages during inflammation, and separate control mechanisms are implicated.
In this study, B cell function in protective TH2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4+ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4+ T cell-mediated protective immunity against N. brasiliensis infection.
Parasitic nematode infections are an extremely important global public health problem. Infections by hookworms and roundworms for example cause anemia, widespread developmental problems and devalued immunity against bacterial infections such as salmonella and tuberculosis. Although treatable with drugs, parasitic nematode re-infections occur as humans do not develop protective immunity. Ultimately, the public health burden caused by these infections will be best controlled by the development of vaccines against nematode infections. For these to be effective, it is important to understand how the various components of the immune system can respond to infection. In this study, we show that B cells, which typically protect against infection by producing antibodies, can also protect against an experimental hookworm like nematode infection by additional mechanisms. This form of protection instead depended on B cells producing cytokines associated with parasitic nematode expulsion and also by providing T cells with specific instruction. Together, these B cell driven responses lead to a rapid resolution of the infection. These important findings indicate that vaccination strategies against nematode parasites such as hookworms need to understand immune responses other than antibody to be optimally protective.
In BALB/c mice, susceptibility to infection with the intracellular parasite Leishmania major is driven largely by the development of T helper 2 (Th2) responses and the production of interleukin (IL)-4 and IL-13, which share a common receptor subunit, the IL-4 receptor alpha chain (IL-4Rα). While IL-4 is the main inducer of Th2 responses, paradoxically, it has been shown that exogenously administered IL-4 can promote dendritic cell (DC) IL-12 production and enhance Th1 development if given early during infection. To further investigate the relevance of biological quantities of IL-4 acting on DCs during in vivo infection, DC specific IL-4Rα deficient (CD11ccreIL-4Rα-/lox) BALB/c mice were generated by gene targeting and site-specific recombination using the cre/loxP system under control of the cd11c locus. DNA, protein, and functional characterization showed abrogated IL-4Rα expression on dendritic cells and alveolar macrophages in CD11ccreIL-4Rα-/lox mice. Following infection with L. major, CD11ccreIL-4Rα-/lox mice became hypersusceptible to disease, presenting earlier and increased footpad swelling, necrosis and parasite burdens, upregulated Th2 cytokine responses and increased type 2 antibody production as well as impaired classical activation of macrophages. Hypersusceptibility in CD11ccreIL-4Rα-/lox mice was accompanied by a striking increase in parasite burdens in peripheral organs such as the spleen, liver, and even the brain. DCs showed increased parasite loads in CD11ccreIL-4Rα-/lox mice and reduced iNOS production. IL-4Rα-deficient DCs produced reduced IL-12 but increased IL-10 due to impaired DC instruction, with increased mRNA expression of IL-23p19 and activin A, cytokines previously implicated in promoting Th2 responses. Together, these data demonstrate that abrogation of IL-4Rα signaling on DCs is severely detrimental to the host, leading to rapid disease progression, and increased survival of parasites in infected DCs due to reduced killing effector functions.
Leishmaniasis is a parasitic infection caused by protozoan parasites of Leishmania species and is transmitted by the sandfly. Disease in humans ranges from localized cutaneous lesions to disseminated visceral Leishmaniasis. Mouse models of Leishmania major infection have demonstrated that a “healing” response in C57BL/6 mice requires the secretion of protective T helper (Th) 1 cytokines, including IFN-γ, which mediates parasite killing by inducing nitric oxide production. Conversely, “non-healer” BALB/c mice are unable to control infection and develop a Th2 immune response characterized by the production of IL-4 and IL-13 cytokines. Although IL-4 is the main inducer of Th2 responses, it has been shown that IL-4 can instruct dendritic cell (DC)-derived IL-12 production and Th1 development if administered during DC activation. To further investigate the role of DCs, a DC specific IL-4Rα-deficient mouse model was established. L. major studies demonstrated hypersusceptibility to infection and strikingly increased parasite loads in peripheral organs of mice lacking IL-4Rα on DCs. Moreover, increased parasite burdens were observed in host cells, including DCs, which showed reduced killing effector functions. In summary, this study demonstrates that IL-4Rα-mediated instruction of DCs occurs in vivo and is necessary to avoid rapid progression of disease in the host.
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.