Interleukin-4 Responsive Dendritic Cells Are Dispensable to Host Resistance Against Leishmania mexicana Infection.

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4-/-, IL-13-/- and IL-4Rα-/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana. Previous studies have ruled out a role for IL-4-mediated protection on CD4+ T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana, since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c+ DCs (CD11ccreIL-4Rα-/lox) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11ccreIL-4Rα-/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11ccreIL-4Rα-/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11ccreIL-4Rα-/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα-/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana. Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection.

Can We Harness Immune Responses to Improve Drug Treatment in Leishmaniasis?

Leishmaniasis is a vector-borne parasitic disease that has been neglected in priority for control and eradication of malaria, tuberculosis, and HIV/AIDS. Collectively, over one seventh of the world's population is at risk of being infected with 0.7-1.2 million new infections reported annually. Clinical manifestations range from self-healing cutaneous lesions to fatal visceral disease. The first anti-leishmanial drugs were introduced in the 1950's and, despite several shortcomings, remain the mainstay for treatment. Regardless of this and the steady increase in infections over the years, particularly among populations of low economic status, research on leishmaniasis remains under funded. This review looks at the drugs currently in clinical use and how they interact with the host immune response. Employing chemoimmunotherapeutic approaches may be one viable alternative to improve the efficacy of novel/existing drugs and extend their lifespan in clinical use.

Unravelling the unsolved paradoxes of cytokine families in host resistance and susceptibility to Leishmania infection.

Leishmaniasis is a neglected disease caused by protozoan parasites of the genus Leishmania. Successful clearance of Leishmania relies on a robust human immune response and various cytokines have been implicated in resistance and susceptibility to Leishmania infection. Accordingly, various immunotherapeutic approaches involving cytokines and cytokine receptors are being considered as novel avenues of treatment given the limited efficacy of current anti-leishmanial drugs. These approaches target canonical T helper (Th)1/Type 1 cytokines as intended mediators of host-protection to infection whilst concomitantly suppressing Th2/Type 2 cytokines and their anticipated disease-promoting roles. However, the use of cytokine and cytokine receptor gene-deficient mice over the years has challenged this simplistic view of Th1/Type 1-mediated resistance and Th2/Type 2-mediated susceptibility. Indeed, contribution to susceptibility vs resistance is only a partial consequence to cytokine action as the overall response is multi-faceted due to the pleiotropic, redundant, antagonistic and synergistic action of cytokines and interactions with immune cells in the diseased state. Notably, while the responses of certain cytokines are selectively host-protective or characteristic disease-enhancers, some ligands exert a response depending on the parasite-species initiating infection. Paradoxically, others play dual or contradictory roles in different Leishmania immunopathologies. Hence, cytokines in disease is an unsolved paradox and a comprehensive knowledge of cytokine interplay is important to guide the development of novel immunotherapeutics against leishmaniasis. In this review, we characterize various cytokine families in persistence and clearance of the Leishmania parasite and particularly elucidate unsolved cytokine puzzles in leishmaniasis based on information acquired from "gain of knowledge by loss of function" studies in cytokine and cytokine receptor gene-deficient mice.

IL-4 Mediated Resistance of BALB/c Mice to Visceral Leishmaniasis Is Independent of IL-4Rα Signaling via T Cells.

Previous studies infecting global IL-4Rα-/-, IL-4-/-, and IL-13-/-mice on a BALB/c background with the visceralizing parasite Leishmania donovani have shown that the T helper 2 cytokines, IL-4, and IL-13, play influential but not completely overlapping roles in controlling primary infection. Subsequently, using macrophage/neutrophil-specific IL-4Rα deficient BALB/c mice, we demonstrated that macrophage/neutrophil unresponsiveness to IL-4 and IL-13 did not have a detrimental effect during L. donovani infection. Here we expand on these findings and show that CD4+ T cell-(Lckcre), as well as pan T cell-(iLckcre) specific IL-4Rα deficient mice, on a BALB/c background, unlike global IL-4Rα deficient mice, are also not adversely affected in terms of resistance to primary infection with L. donovani. Our analysis suggested only a transient and tissue specific impact on disease course due to lack of IL-4Rα on T cells, limited to a reduced hepatic parasite burden at day 30 post-infection. Consequently, the protective role(s) demonstrated for IL-4 and IL-13 during L. donovani infection are mediated by IL-4Rα-responsive cell(s) other than macrophages, neutrophils and T cells.

Inflammatory Dendritic Cells, Regulated by IL-4 Receptor Alpha Signaling, Control Replication, and Dissemination of Leishmania major in Mice.

Leishmaniasis is a vector-borne disease caused by Leishmania parasites. Macrophages are considered the primary parasite host cell, but dendritic cells (DCs) play a critical role in initiating adaptive immunity and controlling Leishmania infection. Accordingly, our previous study in CD11ccreIL-4Rα-/lox mice, which have impaired IL-4 receptor alpha (IL-4Rα) expression on CD11c+ cells including DCs, confirmed a protective role for IL-4/IL-13-responsive DCs in replication and dissemination of parasites during cutaneous leishmaniasis. However, it was unclear which DC subset/s was executing this function. To investigate this, we infected CD11ccreIL-4Rα-/lox and control mice with L. major GFP+ parasites and identified subsets of infected DCs by flow cytometry. Three days after infection, CD11b+ DCs and CD103+ DCs were the main infected DC subsets in the footpad and draining lymph node, respectively and by 4 weeks post-infection, Ly6C+ and Ly6C- CD11b+ DCs were the main infected DC populations in both the lymph nodes and footpads. Interestingly, Ly6C+CD11b+ inflammatory monocyte-derived DCs but not Ly6C-CD11b+ DCs hosted parasites in the spleen. Importantly, intracellular parasitism was significantly higher in IL-4Rα-deficient DCs. In terms of DC effector function, we found no change in the expression of pattern-recognition receptors (TLR4 and TLR9) nor in expression of the co-stimulatory marker, CD80, but MHCII expression was lower in CD11ccreIL-4Rα-/lox mice at later time-points compared to the controls. Interestingly, in CD11ccreIL-4Rα-/lox mice, which have reduced Th1 responses, CD11b+ DCs had impaired iNOS production, suggesting that DC IL-4Rα expression and NO production is important for controlling parasite numbers and preventing dissemination. Expression of the alternative activation marker arginase was unchanged in CD11b+ DCs in CD11creIL-4Rα-/lox mice compared to littermate controls, but RELM-α was upregulated, suggesting IL-4Rα-independent alternative activation. In summary, L. major parasites may use Ly6C+CD11b+ inflammatory DCs derived from monocytes recruited to infection as "Trojan horses" to migrate to secondary lymphoid organs and peripheral sites, and DC IL-4Rα expression is important for controlling infection.

Deletion of Interleukin-4 Receptor Alpha-Responsive Keratinocytes in BALB/c Mice Does Not Alter Susceptibility to Cutaneous Leishmaniasis.

The skin microenvironment at the site of infection plays a role in the early events that determine protective T helper 1/type 1 immune responses during cutaneous leishmaniasis (CL) infection. During CL in nonhealing BALB/c mice, early interleukin-4 (IL-4) can instruct dendritic cells for protective Th1 immunity. Additionally, keratinocytes, which are the principal cell type in the skin epidermis, have been shown to secrete IL-4 early after Leishmania major infection. Here, we investigated whether IL-4/IL-13 signaling via the common IL-4 receptor alpha chain (IL-4Rα) on keratinocytes contributes to susceptibility during experimental CL. To address this, keratinocyte-specific IL-4Rα-deficient (KRT14cre IL-4Rα-/lox) mice on a BALB/c genetic background were generated by gene targeting and site-specific recombination (Cre/loxP) under the control of the keratinocyte-specific krt14 locus. Following high-dose infection with L. major IL-81 and LV39 promastigotes subcutaneously in the footpad, footpad swelling, parasite burden, IFN-γ/IL-4/IL-13 cytokine production, and type 1 and type 2 antibody responses were similar between KRT14cre IL-4Rα-/lox and littermate control IL-4Rα-/lox BALB/c mice. An intradermal infection with low-dose L. major IL-81 and LV39 promastigotes in the ear showed results in infected KRT14cre IL-4Rα-/lox BALB/c mice similar to those of littermate control IL-4Rα-/lox BALB/c mice, with the exception of a significant decrease observed in parasite burden only at the site of LV39 infection in the ear. Collectively, our results show that autocrine and paracrine signaling of IL-4/IL-13 through the IL-4Rα chain on keratinocytes does not influence the establishment of a nonhealing Th2 immune response in BALB/c mice during L. major infection.

Interleukin-4 Receptor Alpha: From Innate to Adaptive Immunity in Murine Models of Cutaneous Leishmaniasis.

The interleukin (IL)-4 receptor alpha (IL-4Rα), ubiquitously expressed on both innate and adaptive immune cells, controls the signaling of archetypal type 2 immune regulators; IL-4 and IL-13, which elicit their signaling action by the type 1 IL-4Rα/gamma common and/or the type 2 IL-4Rα/IL-13Rα complexes. Global gene-deficient mouse models targeting IL-4, IL-13, or the IL-4Rα chain, followed by the development of conditional mice and generation of important cell-type-specific IL-4Rα-deficient mouse models, were indeed critical to gaining in-depth understanding of detrimental T helper (Th) 2 mechanisms in type 1-controlled diseases. A primary example being cutaneous leishmaniasis, which is caused by the protozoan parasite Leishmania major, among others. The disease is characterized by localized self-healing cutaneous lesions and necrosis for which, currently, not a single vaccine has made it to a stage that can be considered effective. The spectrum of human leishmaniasis belongs to the top 10 infectious diseases according to the World Health Organization. As such, 350 million humans are at risk of infection and disease, with an incidence of 1.5-2 million new cases being reported annually. A major aim of our research is to identify correlates of host protection and evasion, which may aid in vaccine design and therapeutic interventions. In this review, we focus on the immune-regulatory role of the IL-4Rα chain from innate immune responses to the development of beneficial type 1 and detrimental type 2 adaptive immune responses during cutaneous Leishmania infection. We discuss the cell-specific requirements of the IL-4Rα chain on crucial innate immune cells during L. major infection, including, IL-4Rα-responsive skin keratinocytes, macrophages, and neutrophils, as well as dendritic cells (DCs). The latter, contributing to one of the paradigm shifts with respect to the role of IL-4 instructing DCs in vivo, to promote Th1 responses against L. major. Finally, we extend these innate responses and mechanisms to control of adaptive immunity and the effect of IL-4Rα-responsiveness on T and B lymphocytes orchestrating the development of CD4+ Th1/Th2 and B effector 1/B effector 2 B cells in response to L. major infection in the murine host.

IL-4Rα Signaling in Keratinocytes and Early IL-4 Production Are Dispensable for Generating a Curative T Helper 1 Response in Leishmania major-Infected C57BL/6 Mice.

Experimental infection with the protozoan parasite Leishmania major has been extensively used to understand the mechanisms involved in T helper cell differentiation. Following infection, C57BL/6 mice develop a small self-healing cutaneous lesion and they are able to control parasite burden, a process linked to the development of T helper (Th) 1 cells. The local presence of IL-12 has been reported to be critical in driving Th1 cell differentiation. In addition, the early secretion of IL-4 was reported to potentially contribute to Th1 cell differentiation. Following infection with L. major, early keratinocyte-derived IL-4 was suggested to contribute to Th1 cell differentiation. To investigate a putative autocrine role of IL-4 signaling on keratinocytes at the site of infection, we generated C57BL/6 mice deficient for IL-4Rα expression selectively in keratinocytes. Upon infection with L. major, these mice could control their inflammatory lesion and parasite load correlating with the development of Th1 effector cells. These data demonstrate that IL-4 signaling on keratinocytes does not contribute to Th1 cell differentiation. To further investigate the source of IL-4 in the skin during the first days after L. major infection, we used C57BL/6 IL-4 reporter mice allowing the visualization of IL-4 mRNA expression and protein production. These mice were infected with L. major. During the first 3 days after infection, skin IL-4 mRNA expression was observed selectively in mast cells. However, no IL-4 protein production was detectable locally. In addition, early IL-4 blockade locally had no impact on subsequent Th1 cell differentiation and control of the disease. Taken together, the present data rule out a major role for skin IL-4 and keratinocyte IL-4Rα signaling in the development of a Th1 protective immune response following experimental infection with L. major.

IL-4-producing B cells regulate T helper cell dichotomy in type 1- and type 2-controlled diseases.

Interleukin-4 (IL-4)-induced T helper (Th) 2 cells promote susceptibility to the protozoan parasite Leishmania major, while conferring immunity to the intestinal trematode Schistosoma mansoni Here, we report that abrogation of IL-4 receptor alpha (IL-4Rα) signaling on B cells in BALB/c mice (mb1creIL-4Rα-/lox) transformed nonhealer BALB/c to a healer phenotype with an early type 1 and dramatically reduced type 2 immune response and an absence of ulceration and necrosis during cutaneous leishmaniasis. From adoptive reconstitution and mixed bone-marrow chimera studies in B cell-deficient (µMT) mice, we reveal a central role for B cell-derived IL-4 and IL-4Rα in the optimal induction of the susceptible type 2 phenotype to L. major infection. We further demonstrate that the absence of IL-4Rα signaling on B cells exacerbated S. mansoni-induced mortality and pathology in BALB/c mice, due to a diminished type 2 immune response. In both disease models, IL-4Rα-responsive B cells displayed increased IL-4 production as early as day 1 after infection. Together, these results demonstrate that IL-4-producing and IL-4Rα-responsive B cells are critical in regulating and assisting early T helper dichotomy toward Th2 responses, which are detrimental in cutaneous leishmaniasis but beneficial in acute schistosomiasis.

Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target.

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker.

Topical Simvastatin as Host-Directed Therapy against Severity of Cutaneous Leishmaniasis in Mice.

We recently demonstrated that statins mediate protection against intracellular pathogens, Mycobacterium tuberculosis and Listeria monocytogenes in mice. Here, we investigated the immunomodulatory potential of simvastatin as a topical or systemic host-directed drug therapy in controlling inflammatory responses in an experimental mouse model of cutaneous leishmaniasis caused by Leishmania major (LV39). In an ear infection model, topical application of simvastatin directly on established lesions significantly reduced severity of the disease reflected by ear lesion size and ulceration. The host protective effect was further accompanied by decreased parasite burden in the ear and draining lymph nodes in both BALB/c and C57BL/6 mice. Pre-treatment of these mice on a low-fat cholesterol diet and systemic simvastatin also reduced footpad swelling, as well as parasite burdens and ulceration/necrosis in the more robust footpad infection model, demonstrating the prophylactic potential of simvastatin for cutaneous leishmaniasis. Mechanistically, following L. major infection, simvastatin-treated primary macrophages responded with significantly reduced cholesterol levels and increased production of hydrogen peroxide. Furthermore, simvastatin-treated macrophages displayed enhanced phagosome maturation, as revealed by increased LAMP-3 expression in fluorescent microscopy and Western blot analysis. These findings demonstrate that simvastatin treatment enhances host protection against L. major by increasing macrophage phagosome maturation and killing effector functions.

Batf2/Irf1 induces inflammatory responses in classically activated macrophages, lipopolysaccharides, and mycobacterial infection.

Basic leucine zipper transcription factor Batf2 is poorly described, whereas Batf and Batf3 have been shown to play essential roles in dendritic cell, T cell, and B cell development and regulation. Batf2 was drastically induced in IFN-γ-activated classical macrophages (M1) compared with unstimulated or IL-4-activated alternative macrophages (M2). Batf2 knockdown experiments from IFN-γ-activated macrophages and subsequent expression profiling demonstrated important roles for regulation of immune responses, inducing inflammatory and host-protective genes Tnf, Ccl5, and Nos2. Mycobacterium tuberculosis (Beijing strain HN878)-infected macrophages further induced Batf2 and augmented host-protective Batf2-dependent genes, particularly in M1, whose mechanism was suggested to be mediated through both TLR2 and TLR4 by LPS and heat-killed HN878 (HKTB) stimulation experiments. Irf1 binding motif was enriched in the promoters of Batf2-regulated genes. Coimmunoprecipitation study demonstrated Batf2 association with Irf1. Furthermore, Irf1 knockdown showed downregulation of IFN-γ- or LPS/HKTB-activated host-protective genes Tnf, Ccl5, Il12b, and Nos2. Conclusively, Batf2 is an activation marker gene for M1 involved in gene regulation of IFN-γ-activated classical macrophages, as well as LPS/HKTB-induced macrophage stimulation, possibly by Batf2/Irf1 gene induction. Taken together, these results underline the role of Batf2/Irf1 in inducing inflammatory responses in M. tuberculosis infection.

The role of IL-4 and IL-13 in cutaneous Leishmaniasis.

Murine models of Leishmania major infection in the 1980s revealed two distinct, counter-regulatory populations of CD4(+) T helper (Th) cells, delineated Th1 and Th2, and their archetypal cytokines, interferon gamma (IFN-γ) and interleukin (IL)-4/IL-13, which promoted resistance/susceptibility to infection, respectively. However, the introduction of global cytokine-deficient mice in the 1990s revealed pleiotropic immune-regulatory mechanisms of IL-4 and IL-13 that either controlled or exacerbated disease. This undermined the basic premise that IL-4/IL-13 played paramount roles in facilitating a non-healing Th2 response to Leishmania infection and instead suggested that both IL-4 and IL-13-dependent and IL-4/IL-13-independent factors orchestrate disease outcome. The recent characterization of cell-type specific IL-4Rα deficient mice was initiated to help reconcile these observations and dissect the cell-specific effects of IL-4/IL-13 during infection. In this review, we summarize original and recent findings with regard to the role of IL-4 and IL-13 in cutaneous Leishmaniasis. Using the information discerned from various studies and our conditional IL-4Rα gene-deficient mice, we particularly discuss the double-edged sword IL-4 (and in some Leishmania disease models IL-13) in driving a susceptible Th2 response, their immune cell targets that support healing or non-healing responses and their novel role in mediating a Th1 response during disease.

Statin therapy reduces the mycobacterium tuberculosis burden in human macrophages and in mice by enhancing autophagy and phagosome maturation.

BACKGROUND: Statins are cholesterol-lowering drugs, targeting HMG-CoA reductase, thereby reducing the risk of coronary disorders and hypercholesterolemia. However, they also can influence immunologic responses. METHODS: Peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDMs) were isolated from patients with familial hypercholesterolemia (FH) during statin therapy. After infection of cells with Mycobacterium tuberculosis, bacterial burden was determined. In vivo, mice were treated with statins before aerosol-based infection with M. tuberculosis and were monitored for disease progression. RESULTS: PBMCs and MDMs from patients with FH receiving statin therapy were more resistant to M. tuberculosis infection, with reduced bacterial burdens, compared with those of healthy donors. Moreover, statin treatment in experimental murine M. tuberculosis infection studies increased host protection, with reduced lung burdens and improved histopathologic findings. Mechanistically, metabolic rescue experiments demonstrated that statins reduce membrane cholesterol levels, particularly by the mevalonate-isoprenoid arm of the sterol pathway. This promoted phagosomal maturation (EEA-1/Lamp-3) and autophagy (LC3-II), as shown by confocal microscopy and Western blot in macrophages. In addition, inhibitors of phagosome and autophagosome maturation reversed the beneficial effect of statins on bacterial growth. CONCLUSION: These results suggest that statin-mediated reduction in cholesterol levels within phagosomal membranes counteract M. tuberculosis-induced inhibition of phagosomal maturation and promote host-induced autophagy, thereby augmenting host protection against tuberculosis.

Deletion of IL-4 receptor alpha on dendritic cells renders BALB/c mice hypersusceptible to Leishmania major infection.

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 (CD11c(cre)IL-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 CD11c(cre)IL-4Rα(-/lox) mice. Following infection with L. major, CD11c(cre)IL-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 CD11c(cre)IL-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 CD11c(cre)IL-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.

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice.

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 (CD11c(cre)IL-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 CD11c(cre)IL-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.

Allergic airway disease is unaffected by the absence of IL-4Rα-dependent alternatively activated macrophages.

BACKGROUND: Markers of alternatively activated macrophages (AAMs) are upregulated in the lungs of asthmatic patients and in mice with allergic airway disease. AAMs are thought to contribute to the pathogenesis of allergic airway disease by virtue of their decreased NO production and increased production of proline and polyamines, which are important in the synthesis of connective tissues such as collagen. OBJECTIVE: We aimed to define the role of AAMs in the pathogenesis of allergic airway disease. METHODS: The IL-4 receptor alpha (IL-4Rα) gene is genetically abrogated in macrophages in LysM(cre)IL-4Rα(-/lox) mice, which therefore have impaired IL-4/IL-13 activation of AAMs through IL-4R types 1 and 2. Responses of LysM(cre)IL-4Rα(-/lox) mice and IL-4Rα(-/lox) littermate controls were examined in ovalbumin- and house dust mite-induced allergic airway disease. RESULTS: IL-4Rα expression was shown to be efficiently depleted from alveolar macrophages, interstitial macrophages, and CD11b(+)MHCII(+) inflammatory macrophages. Although the expression of markers of AAMs such as Ym-1, arginase and found in inflammatory zone 1 was decreased in macrophages of LysM(cre)IL-4Rα(-/lox) mice in chronic ovalbumin-induced allergic airway disease, airway hyperreactivity, T(H)2 responses, mucus hypersecretion, eosinophil infiltration, and collagen deposition were not significantly reduced. LysM(cre)IL-4Rα(-/lox) mice and littermate controls also developed similar responses in acute ovalbumin- and house dust mite-induced allergic airway disease. CONCLUSION: Our results suggest that the presence of AAMs in allergic airway disease may be only an association, as a result of the increased T(H)2 responses present during disease, and that IL-4Rα-dependent AAMs do not play an important role in the pathology of disease.

Anti-peptide antibodies differentiate between plasmodial lactate dehydrogenases.

Malaria lactate dehydrogenase, a glycolytic enzyme, is a malaria diagnostic target in lateral flow immunochromatographic rapid diagnostic tests. Recombinant Plasmodium yoelii LDH was cloned into the pET-28a vector, expressed and the expressed protein purified from a Ni-NTA affinity matrix. A pan-malarial LDH antibody directed against a common malaria LDH peptide (APGKSDKEWNRDDLL) and two anti-peptide antibodies, each targeting a unique Plasmodium falciparum (LISDAELEAIFDC) and Plasmodium vivax (KITDEEVEGIFDC) LDH peptide were raised in chickens. The antibodies were affinity purified with the appropriate peptide affinity matrix. The affinity purified anti-peptide antibodies detected recombinant P. falciparum, P. vivax and P. yoelii LDH and native P. falciparum and P. yoelii LDH in western blots and immunofluorescence studies. The pan-malarial antibody detected LDH from the three malaria species in western blots. The species-specific anti-peptide antibodies differentiated between P. falciparum and P. vivax LDH. Affinity purified chicken antibodies against recombinant PfLDH, PvLDH and PyLDH proteins each detected the parent and orthologous proteins with similar titers in an ELISA. The study supports an anti-peptide antibody approach to the development of diagnostic reagents.