Dominik Rückerl

Local Macrophage Proliferation, Rather than Recruitment from the Blood, Is a Signature of TH2 Inflammation

Proliferation in situ, rather than immune cell recruitment, drives macrophage expansion in response to parasitic infection. A defining feature of inflammation is the accumulation of innate immune cells in the tissue that are thought to be recruited from the blood. We reveal that a distinct process exists in which tissue macrophages undergo rapid in situ proliferation in order to increase population density. This inflammatory mechanism occurred during T helper 2 (TH2)–related pathologies under the control of the archetypal TH2 cytokine interleukin-4 (IL-4) and was a fundamental component of TH2 inflammation because exogenous IL-4 was sufficient to drive accumulation of tissue macrophages through self-renewal. Thus, expansion of innate cells necessary for pathogen control or wound repair can occur without recruitment of potentially tissue-destructive inflammatory cells.

The IL-27 Receptor Chain WSX-1 Differentially Regulates Antibacterial Immunity and Survival during Experimental Tuberculosis

IL-12 is a potent inducer of IFN-γ production and promotes a protective cell-mediated immune response after Mycobacterium tuberculosis infection. Recently, the IL-12-related cytokine IL-27 was discovered, and WSX-1 was identified as one component of the IL-27R complex. To determine the functional significance of IL-27/WSX-1 during tuberculosis, we analyzed the course of infection and the immune response in WSX-1-KO mice after aerosol infection with M. tuberculosis. In the absence of WSX-1, an increased production of the proinflammatory cytokines TNF and IL-12p40 resulted in elevated CD4+ T cell activation and IFN-γ production, which enhanced macrophage effector functions and reduced bacterial loads. This is the first occasion of a selectively gene-deficient mouse strain showing higher levels of protective immunity against M. tuberculosis infection than wild-type mice. However, a concomitantly increased chronic inflammatory response also accelerated death of infected WSX-1-KO mice. In vitro, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation. In conclusion, IL-27 both prevents optimal antimycobacterial protection and limits the pathological sequelae of chronic inflammation.

IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1.

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.

Suppressor of cytokine signaling (SOCS)1 is a key determinant of differential macrophage activation and function

Macrophages become activated by their environment and develop polarized functions: classically activated (M1) macrophages eliminate pathogens but can cause tissue injury, whereas alternatively activated (M2) macrophages promote healing and repair. Mechanisms directing polarized activation, especially in vivo, are not understood completely, and here, we examined the role of SOCS proteins. M2 macrophages activated in vitro or elicited by implanting mice i.p. with the parasitic nematode Brugia malayi display a selective and IL‐4‐dependent up‐regulation of SOCS1 but not SOCS3. Using siRNA‐targeted knockdown in BMDM, we reveal that the enhanced SOCS1 is crucial for IL‐4‐induced M2 characteristics, including a high arginase I:iNOS activity ratio, suppression of T cell proliferation, attenuated responses to IFN‐γ/LPS, and curtailed SOCS3 expression. Importantly, SOCS1 was essential in sustaining the enhanced PI3K activity that drives M2 activation, defining a new regulatory mechanism by which SOCS1 controls M2 polarization. By contrast, for M1 macrophages, SOCS1 was not only an important regulator of proinflammatory mediators (IL‐6, IL‐12, MHC class II, NO), but critically, for M1, we show that SOCS1 also restricted IL‐10 secretion and arginase I activity, which otherwise would limit the efficiency of M1 macrophage proinflammatory responses. Together, our results uncover SOCS1, not only as a feedback inhibitor of inflammation but also as a critical molecular switch that tunes key signaling pathways to effectively program different sides of the macrophage balance.

Alternatively activated macrophages derived from monocytes and tissue macrophages are phenotypically and functionally distinct

Macrophages adopt an alternatively activated phenotype (AAMs) when activated by the interleukin-4receptor(R)α. AAMs can be derived either from proliferation of tissue resident macrophages or recruited inflammatory monocytes, but it is not known whether these different sources generate AAMs that are phenotypically and functionally distinct. By transcriptional profiling analysis, we show here that, although both monocyte and tissue-derived AAMs expressed high levels of Arg1, Chi3l3, and Retnla, only monocyte-derived AAMs up-regulated Raldh2 and PD-L2. Monocyte-derived AAMs were also CX3CR1-green fluorescent protein (GFP)(high) and expressed CD206, whereas tissue-derived AAMs were CX3CR1-GFP and CD206 negative. Monocyte-derived AAMs had high levels of aldehyde dehydrogenase activity and promoted the differentiation of FoxP3(+) cells from naïve CD4(+) cells via production of retinoic acid. In contrast, tissue-derived AAMs expressed high levels of uncoupling protein 1. Hence monocyte-derived AAM have properties associated with immune regulation, and the different physiological properties associated with AAM function may depend on the distinct lineage of these cells.

Induction of IL-4Rα–dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4–driven murine macrophage proliferation in vivo

Macrophage (MΦ) activation must be tightly controlled to preclude overzealous responses that cause self-damage. MicroRNAs promote classical MΦ activation by blocking antiinflammatory signals and transcription factors but also can prevent excessive TLR signaling. In contrast, the microRNA profile associated with alternatively activated MΦ and their role in regulating wound healing or antihelminthic responses has not been described. By using an in vivo model of alternative activation in which adult Brugia malayi nematodes are implanted surgically in the peritoneal cavity of mice, we identified differential expression of miR-125b-5p, miR-146a-5p, miR-199b-5p, and miR-378-3p in helminth-induced MΦ. In vitro experiments demonstrated that miR-378-3p was specifically induced by IL-4 and revealed the IL-4-receptor/PI3K/Akt-signaling pathway as a target. Chemical inhibition of this pathway showed that intact Akt signaling is an important enhancement factor for alternative activation in vitro and in vivo and is essential for IL-4-driven MΦ proliferation in vivo. Thus, identification of miR-378-3p as an IL-4Rα-induced microRNA led to the discovery that Akt regulates the newly discovered mechanism of IL-4-driven macrophage proliferation. Together, the data suggest that negative regulation of Akt signaling via microRNAs might play a central role in limiting MΦ expansion and alternative activation during type 2 inflammatory settings.

Chitinase-like proteins promote IL-17-mediated neutrophilia in a tradeoff between nematode killing and host damage.

Enzymatically inactive chitinase-like proteins (CLPs) such as BRP-39, Ym1 and Ym2 are established markers of immune activation and pathology, yet their functions are essentially unknown. We found that Ym1 and Ym2 induced the accumulation of neutrophils through the expansion of γδ T cell populations that produced interleukin 17 (IL-17). While BRP-39 did not influence neutrophilia, it was required for IL-17 production in γδ T cells, which suggested that regulation of IL-17 is an inherent feature of mouse CLPs. Analysis of a nematode infection model, in which the parasite migrates through the lungs, revealed that the IL-17 and neutrophilic inflammation induced by Ym1 limited parasite survival but at the cost of enhanced lung injury. Our studies describe effector functions of CLPs consistent with innate host defense traits of the chitinase family.

IL-4-producing ILC2s are required for the differentiation of TH2 cells following Heligmosomoides polygyrus infection

Immunity to many human and murine gastrointestinal helminth parasites requires interleukin-4 (IL-4)-directed type 2 helper (TH2) differentiation of CD4+ T cells to elicit type-2 immunity. Despite a good understanding of the inflammatory cascade elicited following helminth infection, the initial source of IL-4 is unclear. Previous studies using the rat helminth parasite Nippostronglyus brasiliensis, identified an important role for basophil-derived IL-4 for TH2 differentiation. However, basophils are redundant for TH2 differentiation following infection with the natural helminth parasite of mice Heligmosomoides polygyrus, indicating that other sources of IL-4 are required. In this study using H. polygyrus, which is controlled by IL-4-dependent immunity, we identified that group-2 innate lymphoid cells (ILC2s) produced significant amounts of IL-4 and IL-2 following H. polygyrus infection. Leukotriene D4 was sufficient to stimulate IL-4 secretion by ILC2s, and the supernatant from activated ILC2s could potently drive TH2 differentiation in vitro in an IL-4-dependent manner. Furthermore, specific deletion of IL-4 from ILC2s compromised TH2 differentiation in vivo. Overall, this study highlights a previously unrecognized and important role for ILC2-derived IL-4 for TH2 differentiation in a natural TH2-dependent model of human helminthiasis.

Macrophage proliferation, provenance, and plasticity in macroparasite infection

Macrophages have long been center stage in the host response to microbial infection, but only in the past 10–15 years has there been a growing appreciation for their role in helminth infection and the associated type 2 response. Through the actions of the IL‐4 receptor α (IL‐4Rα), type 2 cytokines result in the accumulation of macrophages with a distinctive activation phenotype. Although our knowledge of IL‐4Rα‐induced genes is growing rapidly, the specific functions of these macrophages have yet to be established in most disease settings. Understanding the interplay between IL‐4Rα‐activated macrophages and the other cellular players is confounded by the enormous transcriptional heterogeneity within the macrophage population and by their highly plastic nature. Another level of complexity is added by the new knowledge that tissue macrophages can be derived either from a resident prenatal population or from blood monocyte recruitment and that IL‐4 can increase macrophage numbers through proliferative expansion. Here, we review current knowledge on the contribution of macrophages to helminth killing and wound repair, with specific attention paid to distinct cellular origins and plasticity potential.

Dendritic Cell-Derived IL-12p40 Homodimer Contributes to Susceptibility in Cutaneous Leishmaniasis in BALB/c Mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1αβ, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released ∼2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1αβ, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.