Christoph Hölscher

Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways

Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit+ erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. These precursors developed into CD45+ c-kitlo CX3CR1− immature (A1) cells and matured into CD45+ c-kit− CX3CR1+ (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia.

Cutting Edge: Toll-Like Receptor (TLR)2- and TLR4-Mediated Pathogen Recognition in Resistance to Airborne Infection with Mycobacterium tuberculosis

Innate resistance against Mycobacterium tuberculosis is thought to depend critically on engagement of pattern recognition receptors on macrophages. However, the relative contribution of these receptors for containing M. tuberculosis infection has remained unexplored in vivo. To address this issue, we infected mice defective in CD14, TLR2, or TLR4 with M. tuberculosis by aerosol. Following infection with 100 mycobacteria, either mutant strain was as resistant as congenic control mice. Granuloma formation, macrophage activation, and secretion of proinflammatory cytokines in response to low-dose aerosol infection were identical in mutant and control mice. However, high-dose aerosol challenge with 2000 CFU M. tuberculosis revealed TLR2-, but not TLR4-defective mice to be more susceptible than control mice. In conclusion, while TLR2 signaling contributes to innate resistance against M. tuberculosis in borderline situations, its function, and that of CD14 and TLR4, in initiating protective responses against naturally low-dose airborne infection is redundant.

Regulated Expression of Nuclear Receptor RORγt Confers Distinct Functional Fates to NK Cell Receptor-Expressing RORγt+ Innate Lymphocytes

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.

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.

Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRγ–Syk–Card9–dependent innate immune activation

Novel vaccination strategies against Mycobacterium tuberculosis (MTB) are urgently needed. The use of recombinant MTB antigens as subunit vaccines is a promising approach, but requires adjuvants that activate antigen-presenting cells (APCs) for elicitation of protective immunity. The mycobacterial cord factor Trehalose-6,6-dimycolate (TDM) and its synthetic analogue Trehalose-6,6-dibehenate (TDB) are effective adjuvants in combination with MTB subunit vaccine candidates in mice. However, it is unknown which signaling pathways they engage in APCs and how these pathways are coupled to the adaptive immune response. Here, we demonstrate that these glycolipids activate macrophages and dendritic cells (DCs) via Syk–Card9–Bcl10–Malt1 signaling to induce a specific innate activation program distinct from the response to Toll-like receptor (TLR) ligands. APC activation by TDB and TDM was independent of the C-type lectin receptor Dectin-1, but required the immunoreceptor tyrosine-based activation motif–bearing adaptor protein Fc receptor γ chain (FcRγ). In vivo, TDB and TDM adjuvant activity induced robust combined T helper (Th)-1 and Th-17 T cell responses to a MTB subunit vaccine and partial protection against MTB challenge in a Card9-dependent manner. These data provide a molecular basis for the immunostimulatory activity of TDB and TDM and identify the Syk–Card9 pathway as a rational target for vaccine development against tuberculosis.

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4+ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.

Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum

The obligate intracellular pathogen Leishmania major survives and multiplies in professional phagocytes. The evasion strategy to circumvent killing by host phagocytes and establish a productive infection is poorly understood. Here we report that the virulent inoculum of Leishmania promastigotes contains a high ratio of annexin A5-binding apoptotic parasites. This subpopulation of parasites is characterized by a round body shape, a swollen kinetoplast, nuclear condensation, and a lack of multiplication and represents dying or already dead parasites. After depleting the apoptotic parasites from a virulent population, Leishmania do not survive in phagocytes in vitro and lose their disease-inducing ability in vivo. TGF-β induced by apoptotic parasites is likely to mediate the silencing of phagocytes and lead to survival of infectious Leishmania populations. The data demonstrate that apoptotic promastigotes, in an altruistic way, enable the intracellular survival of the viable parasites.

Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke

The devastating effect of ischemic stroke is attenuated in mice lacking conventional and unconventional T cells, suggesting that inflammation enhances tissue damage in cerebral ischemia. We explored the functional role of αβ and γδ T cells in a murine model of stroke and distinguished 2 different T cell-dependent proinflammatory pathways in ischemia-reperfusion injury. IFN-γ produced by CD4(+) T cells induced TNF-α production in macrophages, whereas IL-17A secreted by γδ T cells led to neutrophil recruitment. The synergistic effect of TNF-α and IL-17A on astrocytes resulted in enhanced secretion of CXCL-1, a neutrophil chemoattractant. Application of an IL-17A-blocking antibody within 3 hours after stroke induction decreased infarct size and improved neurologic outcome in the murine model. In autoptic brain tissue of patients who had a stroke, we detected IL-17A-positive lymphocytes, suggesting that this aspect of the inflammatory cascade is also relevant in the human brain. We propose that selective targeting of IL-17A signaling might provide a new therapeutic option for the treatment of stroke.

The Lymphotoxin β Receptor Is Critically Involved in Controlling Infections with the Intracellular Pathogens Mycobacterium tuberculosis and Listeria monocytogenes

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-γ and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LTβR pathway. When mice deficient in LTα or LTβ were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LTαβ heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LTα1β2 heterotrimers (LTβR-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LTβR-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LTβR-KO mice had similar transcript levels of TNF and IFN-γ and recruited similar numbers of CD3+ T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LTβR is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LTβR-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LTα1β2 heterotrimers via the LTβR is an essential prerequisite for containment of intracellular pathogens.

Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9.

The role of Toll‐like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR‐mediated pathogen recognition and MyD88‐dependent signaling events on anti‐mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple‐ and MyD88‐deficient mice. After aerosol infection, both TLR2/4/9‐deficient and wild‐type mice expressed pro‐inflammatory cytokines promoting antigen‐specific T cells and the production of IFN‐γ to similar extents. Moreover, TLR2/4/9‐deficient mice expressed IFN‐γ‐dependent inducible nitric oxide synthase and LRG‐47 in infected lungs. MyD88‐deficient mice expressed pro‐inflammatory cytokines and were shown to expand IFN‐γ‐producing antigen‐specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9‐deficient mice controlled Mtb replication. IFN‐γ‐dependent restriction of mycobacterial growth was severely impaired only in Mtb‐infected MyD88, but not in TLR2/4/9‐deficient bone marrow‐derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, ‐4, and ‐9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti‐mycobacterial defense.