IFN-γ Signals a Changing of the Guards

Abstract

Following acute inflammation or infection, tissue-resident macrophages are replaced by inflammatory DCs and effector macrophages that arise from circulating monocytes. In this issue of Immunity, Goldszmid et al. (2012) demonstrate a central role for NK cell-derived IFN-γ in signaling and facilitating this transition.

Inflammation--initiated by either sterile or infectious stimuli-- is associated with the recruitment of circulating CCR2-dependent monocytes to the site of tissue injury and infection (Ingersoll et al., 2011). In the spleen of Listeria-infected mice, recruited CCR2-dependent monocytes have been shown to differentiate into tumor necrosis factor (TNF) and inducible NO synthase (iNOS)-producing dendritic cells(DCs) or so-called Tip DCs, which exhibit potent antimicrobial effector functions against bacterial infection(Serbina et al., 2003). Cells resembling TipDCs have been observed during experimental infections using viral (influenza, herpes simplex virus-2), bacterial (Salmonella, Brucella melitensis), fungal (Aspergillus fumigatus and Blastomyces dermatitidis) and parasitic (Leishmania major and Toxoplasma gondii) agents. Following oral inoculation of T. gondii cysts, CCR2-dependent monocytes are rapidly recruited to the villi of the small intestine where the excysted parasites initiate the infectious process. Inflammatory monocytes differentiate-presumably in response to local parasite and tissue–derived factors- into F4/80⁺ Ly6C^hi but CD11c^lomacrophages with the ability to express TNF and iNOS and mediate restriction of Toxoplasma replication in the gut(Dunay et al., 2008). In addition to mediating antimicrobial resistance, inflammatory monocytes have also been shown to differentiate into classical CD11c⁺ dendritic cells. In Leishmania major infection, monocyte-derived DCs migrate from the skin and contribute to the generation of protective Th1 cells in the lymph node(Leon et al., 2007). In contrast, these cells are dispensable for the priming of Th1 cell responses to HSV-2, but are crucial for restimulation of effector Th1 cells in the vaginal mucosa(Iijima et al., 2011). Although the roles of CCR2 signaling chemokines in regulating monocyte egress from the bone marrow and their subsequent recruitment into inflamed tissues have been investigated, the microenvironmental signals driving monocyte differentiation into antimicrobial effector cells or Th1 cell-promoting DCs have remained elusive. Now, Goldzmid et al. (2012), identify a local positive feedback mechanism that promotes the differentiation of migratory monocytes into DCs expressing IL-12. They identify IFN-γ--the very end product of the Th1 cell response cascade-- as the critical inducer of the inflammatory DC phenotype. Surprisingly, IFN-γ signaling is, itself required for the attrition of the resident macrophage population and thus serves as a switch for the tissue transitioning from a homeostatic to an inflammatory state (Figure1).

Figure 1. NK Cell -Derived IFN-γ Signals Removal of Resident Macrophages and Local Differentiation of Recruited Monocytes into IL-12 producing Inflammatory DCs and Effector Macrophages.

LyC6⁺ CCR2⁺inflammatory monocytes are recruited to the peritoneal cavity of mice infected with Toxoplasma cysts and differentiate into inflammatory macrophages and monocyte-derived DCs. Mo-DCs are the main source of IL-12 at the site of infection and require IFN-γ and MyD88 signals for IL-12 production. IFN-γ, derived from NK cells, is also required for the removal of resident macrophages and the expression of macrophage and DC maturation markers in monocyte-derived cells. As the main producers of IL-12, Mo-DCs might be important for the priming and/or restimulation of Th1 cell responses at the infection site.

Given the centrality of IL-12 in driving protective Th1 and CD8⁺T cell effector responses to T. gondii, the authors started their work by focusing on the identity of the IL-12 producing cell at the site of T. gondii inoculation. Prior studies using in vivo injections of a soluble extract of the parasite have identified CD8α⁺ DCs in the T cell zone as spleen-resident innate cells mounting an immediate IL-12 response(Reis e Sousa et al., 1997). Indeed, with the recent studies of T. gondii infection in the Batf3-deficient mice (which lack CD8α⁺ and CD103⁺ DC subsets), the critical role of CD8α⁺ DCs in producing the initial burst of IL-12 required for priming Th1 cell responses to T. gondii has now been affirmed (Mashayekhi et al., 2011). However, the nature of the IL-12-producing cell present at the infection site, where effector Th1 cell responses are needed to restrain parasite replication, is not known. To address this issue, the authors took advantage of a previously constructed IL-12p40 reporter mouse strain and have utilized an intraperitoneal route of inoculation of intact parasite cysts. Different from the natural peroral route, this experimental system allowed them to sample the infection site and characterize the phenotype of the IL-12-producing cells present in the peritoneal cavity, without need for tissue digestion, cell purification and, importantly, without the additional confounding effects of innate cell exposure to the encroachinggut microbiome. Adoptive transfer of monocytes isolated from the bone marrow of the IL-12p40 reporter strain also allowed tracking and detailed analysis of how acquisition of the IL-12- producing phenotype is controlled as recruited precursors differentiate into inflammatory DCs.

Based on these approaches, Goldzmid et al. make several important observations and tantalizing insights. First, IL-12 expression was restricted to F4/80⁺ CD8α-inflammatory DCs whereas resident macrophages remained IL-12 negative and became superceded by the influx of neutrophils, inflammatory macrophages and DCs. The authors subsequently investigated the monocyte-autonomous roles of IFN-γ and MyD88 signaling. They find that both are required for IL-12 production by inflammatory DCs, This result is, on its surface, predictable, given the documented role of TLR11 in innate sensing of T. gondii and the well-known priming effect of IFN-γ for the transcriptional activation of the IL-12p40 gene. However, the finding that ablation of IFN-γ signaling results in a more severe curtailment of maturation and IL-12 production is unanticipated. In the absence of IFN-γ, recruited monocytes still differentiated into CD11c⁺DCs but did not exhibit the mature Ly6C^hi F4/80⁺IL-12⁺ inflammatory phenotype. This dissociation indicates additional growth factors or cytokines provide differentiative signals distinct from the maturational effects of IFN-γ and TLR engagement. Thus, depending upon which complement of hemopoietic growth factors the recruited monocytes encounter, they might differentiate into either macrophages or DCs. Subsequently, synergistic activation by endogenous IFN-γ and pathogen-derived ligands could then represent a shared pathway for arming and licensing macrophages and DCs into antimicrobial effectors and pro-inflammatory antigen presenting cells (APCs).

A most fascinating aspect of this work relates to the behavior of the resident macrophage population that first encounters T. gondii. Why these cells fail to mount an IL-12 response remains a mystery. It is possible that they do not express the requisite components (eg., TLR 11, Irf8) essential for pathogen-sensing and transcription of the IL-12 genes. Apparently, IFN-γ exposure of these pre-existing sentinels does not trigger the same maturational effects observed in the migrant-derived DC and macrophage populations. Instead of activating resident macrophages, IFN-γ initiates a cascade of events leading to their attrition. The IFN-γ-dependent mechanisms responsible for their disappearance are unclear and need to be worked out. Whether resident macrophages are direct targets of IFN-γ signaling and the modality of resident macrophage cell death are two outstanding issues that require further probing.

To provide a glimpse into the maturational program initiated by IFN-γ signaling resulting in IL-12 production and acquisition of antigen presenting function by inflammatory DCs, Goldzmid et al., compared the gene expression profiles of sorted CD11c+ inflammatory DCs obtained from infected mice under IFN-γ sufficient and deficient conditions. Consistent with their phenotypic analysis, neutralization of IFN-γ in vivo led to suppression of only a limited subset of genes necessary for antigen presentation and IL-12 production. Whereas DC expression of Ciita, Ccr7 and Cd70 was IFNγ-independent, IFN-γ blockade led to suppression of Cd86 and Irf8 expression. Given that Irf8 is essential for the development of IL-12 producing CD8α+ DCs(Aliberti et al., 2003), the principal effect of IFN-γ signaling could be to activate the latent Th1 cell promoting potential of inflammatory DC precursors, through induction of Irf8 and, potentially, T-bet. The current work leaves open questions regarding the relative importance of these inflammatory IL-12⁺ DCs in priming versus subsequent restimulation of Th1 cells in the spleen and at the peripheral effector site. It could well be that, in the case of T. gondii, there is a division of labor amongst DC subsets, with CD8α⁺ DCs serving as the priming APC in the spleen and the monocyte-derived inflammatory DCs acting as principal APCs in the periphery. As alluded to above, the contribution of inflammatory DCs to the initial priming of T cells will probably vary depending upon factors inherent to the biology of the infectious agent and its route of invasion or transmission, perhaps with an enhanced prominence of the inflammatory DC’s priming roleat mucosal sites, such as the lung(Hohl et al., 2009).

Viewed from a broader perspective, the finding that NK cell-derived IFN-γ plays a key role in programming the Th1 cell-promoting functional module within inflammatory DCs suggests a general paradigm for how early production of the archetypal Th1 and Th2 cell-type cytokines by innate lymphoid cells, conditions the development of migrant monocytes into antigen presenting cell type required for subsequent induction or amplification of the appropriately matched adaptive immune responses.