A Myeloid-Specific Lack of IL-4Rα Prevents the Development of Alternatively Activated Macrophages and Enhances Immunity to Experimental Cysticercosis.

To determine the role that the IL-4/IL13 receptor plays in the development of alternatively activated macrophages (AAM or M2) and their role in the regulation of immunity to the extraintestinal phase of the helminth parasite Taenia crassiceps, we followed the infection in a mouse strain lacking the IL-4Rα gene (IL-4Rα-/-) and in the macrophage/neutrophil-specific IL-4Rα-deficient mouse strain (LysMcreIL-4Rα-/lox or cre/LoxP). While 100% of T. crassiceps-infected IL-4Rα+/+ (WT) mice harbored large parasite loads, more than 50% of th eIL-4Rα-/- mice resolved the infection. Approximately 88% of the LysMcreIL-4Rα-/lox mice displayed a sterilizing immunity to the infection. The remaining few infected cre/LoxP mice displayed the lowest number of larvae in their peritoneal cavity. The inability of the WT mice to control the infection was associated with antigen-specific Th2-type responses with higher levels of IgG1, IL-4, IL-13, and total IgE, reduced NO production, and increased arginase activity. In contrast, IL-4Rα-/- semi-resistant mice showed a Th1/Th2 combined response. Furthermore, macrophages from the WT mice displayed higher transcripts for Arginase-1 and RELM-α, as well as increased expression of PD-L2 with robust suppressive activity over anti-CD3/CD28 stimulated T cells; all of these features are associated with the AAM or M2 macrophage phenotype. In contrast, both the IL-4Rα-/- and LysMcreIL-4Rα-/lox mice did not fully develop AAM or display suppressive activity over CD3/CD28 stimulated T cells, reducing PDL2 expression. Additionally, T-CD8+ but no T-CD4+ cells showed a suppressive phenotype with increased Tim-3 and PD1 expression in WT and IL-4Rα-/-, which were absent in T. crassiceps-infected LysMcreIL-4Rα-/lox mice. These findings demonstrate a critical role for the IL-4 signaling pathway in sustaining AAM and its suppressive activity during cysticercosis, suggesting a pivotal role for AAM in favoring susceptibility to T. crassiceps infection. Thus, the absence of these suppressor cells is one of the leading mechanisms to control experimental cysticercosis successfully.

PKCδ regulates IL-12p40/p70 production by macrophages and dendritic cells, driving a type 1 healer phenotype in cutaneous leishmaniasis.

The protein kinase C (PKC) family is involved in the regulation of many intracellular signalling pathways. Here, we report that the PKCδ isoform regulates IL-12p40/p70 production in macrophages and DC and that PKCδ deficiency in mice transforms the 129/Sv healer to a non-healer strain during cutaneous leishmaniasis. Leishmania major-infected PKCδ(-/-) 129/Sv mice developed a rapid increase in footpad swelling and parasite burden with disease progression, leading to necrosis and ulceration similar to non-healer BALB/c mice. Moreover, PKCδ(-/-) mice failed to develop delayed-type hypersensitivity responses against Leishmania antigen. PKCδ(-/-) macrophages were fully functional with normal MHC class II surface expression and GM-CSF production, recruitment to the draining lymph node and killing effector functions by NO production. In contrast, macrophages and DC produced significantly reduced IL-12p40 and IL-12p70 compared to the WT cells. Decreased IL-12 production resulted in diminished Th1 differentiation, as determined by a striking reduction in IFN-γ by antigen-specific stimulated CD4(+) T cells isolated from popliteal lymph nodes of L. major-infected PKCδ(-/-) mice, explaining the "non-healer" phenotype. We conclude from these data that PKCδ is a regulator of IL-12p40/p70 production by DC and macrophages, driving the healer phenotype during cutaneous leishmaniasis.

Analyzing classical and alternative macrophage activation in macrophage/neutrophil-specific IL-4 receptor-alpha-deficient mice.

Macrophage activation can be divided into a classical and an alternative pathway. Interferon-gamma-induced, classically activated macrophages are indispensable for protective effector responses against intracellular pathogens. However, excessive inflammatory immune responses mediated by classical macrophage activation can also be detrimental to the host. In contrast, the IL-4 receptor-alpha-mediated alternative pathway of macrophage activation has been proposed as a mechanism to attenuate excessive inflammation. Indeed, the generation of macrophage/neutrophil-specific IL-4 receptor-alpha-deficient mice (LysMcreIL-4Ralphaalpha-/lox) enables us now to evaluate the importance of this type of macrophage activation in vivo. Thus, the analysis of LysMcreIL-4Ralpha-/lox mice and the phenotypic characterization of macrophage activation during inflammatory immune responses become of major importance for inflammation research, and useful markers have been identified that allow classically and alternatively activated macrophages to be distinguished. Inducible nitric oxide synthase and arginase-1 are not only prototypical markers of classical and alternative macrophage activation, but both enzymes are also strongly involved in regulating macrophage effector mechanisms and inflammatory immune responses. In this chapter, we describe the use of LysMcreIL-4Ralpha-/lox mice and present experimental procedures to determine classical versus alternative macrophage activation by analyzing nitric oxide synthase and arginase-1 in vitro and in vivo in this murine model.

Interleukin-4-promoted T helper 2 responses enhance Nippostrongylus brasiliensis-induced pulmonary pathology.

The role of CD4(+) T-cell interleukin-4 (IL-4) receptor alpha (IL-4Ralpha) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4(+) T-cell IL-4Ralpha 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-4Ralpha KO mice, the associated lung pathology was reduced. Infected CD4(+) T-cell IL-4Ralpha KO mice showed abrogation of airway mucus production. Furthermore, CD4(+) T-cell IL-4Ralpha 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-4Ralpha 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-4Ralpha KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4(+) T-cell IL-4Ralpha 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.

Murine IL-4 is able to signal via chimeric human IL-4Ralpha/mouse gamma-chain receptor.

Human IL-4Ralpha binds to mouse gammac resulting in a chimeric receptor specific for human IL-4 but not mouse IL-4, providing in principle an inducible hIL-4 system. We investigated the in vitro and in vivo characteristics of human IL-4Ralpha transgenic mice on a mouse IL-4Ralpha-deficient background (hIL-4Ralpha Tg/mIL-4Ralpha(-/-)). The integrity of lymphocyte-specific hIL-4Ralpha expression in hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice was demonstrated by FACS analysis. This was confirmed in functional studies as lymphocytes responded to recombinant hIL-4 but not mIL-4 or mIL-13 in proliferation and T helper differentiation assays, demonstrating species-specificity and inducibility of the chimeric receptor in vitro. We then infected transgenic mice with Nippostrongylus brasiliensis, known to induce a strong Type 2 response in wild-type mice. As expected hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice were unable to expel N. brasiliensis worms which confirms unresponsiveness in non-lymphocytes. However they developed a Th2 cytokine and IgE response in the absence of induction with hIL-4. These results suggested that lymphocyte-specific IL-4Ralpha responsiveness was still present in vivo. Neutralization of endogenous mIL-4 resulted in inhibition of N. brasiliensis-induced Th2 cytokine and total IgE production in hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice suggesting that mIL-4 was involved. Intercrossing hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice with mIL-4(-/-)/mIL-13(-/-) mice completely abrogated Type 2 responses in N. brasiliensis infections. Together, these data demonstrate that mIL-4 triggered the hIL-4Ralpha/mgammac chimeric receptor in vivo.

Protein kinase C delta is essential for optimal macrophage-mediated phagosomal containment of Listeria monocytogenes.

Activation of macrophages and subsequent "killing" effector functions against infectious pathogens are essential for the establishment of protective immunity. NF-IL6 is a transcription factor downstream of IFN-gamma and TNF in the macrophage activation pathway required for bacterial killing. Comparison of microarray expression profiles of Listeria monocytogenes (LM)-infected macrophages from WT and NF-IL6-deficient mice enabled us to identify candidate genes downstream of NF-IL6 involved in the unknown pathways of LM killing independent of reactive oxygen intermediates and reactive nitrogen intermediates. One differentially expressed gene, PKCdelta, had higher mRNA levels in the LM-infected NF-IL6-deficient macrophages as compared with WT. To define the role of PKCdelta during listeriosis, we infected PKCdelta-deficient mice with LM. PKCdelta-deficient mice were highly susceptible to LM infection with increased bacterial burden and enhanced histopathology despite enhanced NF-IL6 mRNA expression. Subsequent studies in PKCdelta-deficient macrophages demonstrated that, despite elevated levels of proinflammatory cytokines and NO production, increased escape of LM from the phagosome into the cytoplasm and uncontrolled bacterial growth occurred. Taken together these data identified PKCdelta as a critical factor for confinement of LM within macrophage phagosomes.

Deletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection.

Effector responses induced by polarized CD4+ T helper 2 (Th2) cells drive nonhealing responses in BALB/c mice infected with Leishmania major. Th2 cytokines IL-4 and IL-13 are known susceptibility factors for L. major infection in BALB/c mice and induce their biological functions through a common receptor, the IL-4 receptor alpha chain (IL-4Ralpha). IL-4Ralpha-deficient BALB/c mice, however, remain susceptible to L. major infection, indicating that IL-4/IL-13 may induce protective responses. Therefore, the roles of polarized Th2 CD4+ T cells and IL-4/IL-13 responsiveness of non-CD4+ T cells in inducing non-healer or healer responses have yet to be elucidated. CD4+ T cell-specific IL-4Ralpha (Lck(cre)IL-4Ralpha(-/lox)) deficient BALB/c mice were generated and characterized to elucidate the importance of IL-4Ralpha signaling during cutaneous leishmaniasis in the absence of IL-4-responsive CD4+ T cells. Efficient deletion was confirmed by loss of IL-4Ralpha expression on CD4+ T cells and impaired IL-4-induced CD4+ T cell proliferation and Th2 differentiation. CD8+, gammadelta+, and NK-T cells expressed residual IL-4Ralpha, and representative non-T cell populations maintained IL-4/IL-13 responsiveness. In contrast to IL-4Ralpha(-/lox) BALB/c mice, which developed ulcerating lesions following infection with L. major, Lck(cre)IL-4Ralpha(-/lox) mice were resistant and showed protection to rechallenge, similar to healer C57BL/6 mice. Resistance to L. major in Lck(cre)IL-4Ralpha(-/lox) mice correlated with reduced numbers of IL-10-secreting cells and early IL-12p35 mRNA induction, leading to increased delayed type hypersensitivity responses, interferon-gamma production, and elevated ratios of inducible nitric oxide synthase mRNA/parasite, similar to C57BL/6 mice. These data demonstrate that abrogation of IL-4 signaling in CD4+ T cells is required to transform non-healer BALB/c mice to a healer phenotype. Furthermore, a beneficial role for IL-4Ralpha signaling in L. major infection is revealed in which IL-4/IL-13-responsive non-CD4+ T cells induce protective responses.

Delayed goblet cell hyperplasia, acetylcholine receptor expression, and worm expulsion in SMC-specific IL-4Ralpha-deficient mice.

Interleukin 4 receptor alpha (IL-4Ralpha) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response-driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Ralpha pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Ralpha-deficient mice (SM-MHC(Cre)IL-4Ralpha(-/lox)) were generated and characterized to uncover any role for IL-4/IL-13 in this non-immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Ralpha was absent from alpha-actin-positive smooth muscle cells, while other cell types showed normal IL-4Ralpha expression, thus demonstrating efficient cell-type-specific deletion of the IL-4Ralpha gene. N. brasiliensis-infected SM-MHC(Cre)IL-4Ralpha(-/lox) mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Ralpha-responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions.

Impairment of alternative macrophage activation delays cutaneous leishmaniasis in nonhealing BALB/c mice.

Expressed on various cell types, the IL-4Ralpha is a component of both receptors for IL-4 and IL-13. Susceptibility of BALB/c mice to Leishmania major is believed to be dependent on the development of IL-4- and IL-13-producing Th2 cells, while IFN-gamma secretion by Th1 cells is related to resistance. Despite a sustained development of Th2 cells, IL-4Ralpha-deficient BALB/c mice are able to control acute cutaneous leishmaniasis, suggesting that IL-4Ralpha-bearing cells other than Th2 cells contribute to susceptibility. To analyze the contribution of the IL-4Ralpha on macrophages, recently generated macrophage/neutrophil-specific IL-4Ralpha-deficient mice on a susceptible BALB/c genetic background were infected with L. major. Strikingly, macrophage/neutrophil-specific IL-4Ralpha-deficient mice showed a significantly delayed disease progression with normal Th2 and type 2 Ab responses but improved macrophage leishmanicidal effector functions and reduced arginase activity. Together, these results suggest that alternative macrophage activation contributes to susceptibility in cutaneous leishmaniasis.

IL-9 is a susceptibility factor in Leishmania major infection by promoting detrimental Th2/type 2 responses.

IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.

Development of an anti-IL-12 p40 auto-vaccine: protection in experimental autoimmune encephalomyelitis at the expense of increased sensitivity to infection.

IL-12 and IL-23, which share the IL-12 p40 subunit, have been ascribed central roles in many autoimmune disorders. We describe here an anti-IL-12 (alphaIL-12) auto-vaccine that potentially blocks both factors in vivo. Immunization of mice with mouse IL-12 coupled to OVA or Pan DR epitope (PADRE) peptide induced Ab directed against the IL-12 p40 subunit, which prevented IFN-gamma production in response to IL-12 administration in vivo. Experimental autoimmune encephalomyelitis, an IL-23-dependent disease model, induced in SJL mice with a proteolipid protein (PLP) peptide was almost undetectable after alphaIL-12 vaccination. Myelin oligodendrocyte glycoprotein (MOG)-induced disease in C57BL/6 mice was also significantly inhibited. This protection correlated with inhibited Th1 cytokine responses in vitro and with an increase in the IgG1/IgG2a anti-PLP Ab balance. Detrimental consequences of alphaIL-12 vaccination were evaluated in C57BL/6 mice infected with Leishmania major (L.m.). While delayed-type hypersensitivity (DTH) suppression and immunoglobulin as well as interleukin production patterns reflected a major shift toward a Th2-type response, L.m. growth was still significantly retarded as compared to that seen in susceptible BALB/c mice. However, vaccinated animals ultimately failed to control parasite expansion. These results suggest that some chronic autoimmune diseases may benefit from alphaIL-12 vaccination at the expense of reduced, but not completely abrogated, cell-mediated immunity.

Alternative macrophage activation is essential for survival during schistosomiasis and downmodulates T helper 1 responses and immunopathology.

Macrophage/neutrophil-specific IL-4 receptor alpha-deficient mice (LysM(Cre)IL-4Ralpha(-/flox)) were generated to understand the role of IL-4/IL-13 responsive myeloid cells during Type 2 immune responses. LysM(Cre)IL-4Ralpha(-/flox) mice developed protective immunity against Nippostrongylus brasiliensis accompanied by T(H)2 development and goblet cell hyperplasia. In contrast, LysM(Cre)IL-4Ralpha(-/flox) mice were extremely susceptible to Schistosoma mansoni infection with 100% mortality during acute infection. Mortality was not dependent on neutrophils and occurred in the presence of T(H)2/Type 2 responses, granuloma formation, and egg-induced fibrosis. Death was associated with increased T(H)1 cytokines, hepatic and intestinal histopathology, increased NOS-2 activity, impaired egg expulsion, and sepsis. IL-10 was not able to compensate for the absence of IL-4/IL-13-activated alternative macrophages. Together, this shows that alternative macrophages are essential during schistosomiasis for protection against organ injury through downregulation of egg-induced inflammation.