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Summary Paneth cells are the primary source of C-type lysozyme, a β-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn’s disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1−/− hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Summary Paneth cells are post-mitotic intestinal epithelial cells supporting the stem cell niche and mucosal immunity. Paneth cell pathologies are observed in various gastrointestinal diseases, but their plasticity and response to genomic and environmental challenges remain unclear. Using a knockin allele engineered at the mouse Lyz1 locus, we performed detailed Paneth cell-lineage tracing. Irradiation induced a subset of Paneth cells to proliferate and differentiate into villus epithelial cells. RNA sequencing (RNA-seq) revealed that Paneth cells sorted from irradiated mice acquired a stem cell-like transcriptome; when cultured in vitro, these individual Paneth cells formed organoids. Irradiation activated Notch signaling, and forced expression of Notch intracellular domain (NICD) in Paneth cells, but not Wnt/β-catenin pathway activation, induced their dedifferentiation. This study documents Paneth cell plasticity, particularly their ability to participate in epithelial replenishment following stem cell loss, adding to a growing body of knowledge detailing the molecular pathways controlling injury-induced regeneration.

Apicomplexan protozoan pathogens avoid destruction and establish a replicative niche within host cells by forming a nonfusogenic parasitophorous vacuole (PV). Here we present evidence for lysosome-mediated degradation of Toxoplasma gondii after invasion of macrophages activated in vivo. Pathogen elimination was dependent on the interferon γ inducible-p47 GTPase, IGTP, required PI3K activity, and was preceded by PV membrane indentation, vesiculation, disruption, and, surprisingly, stripping of the parasite plasma membrane. Denuded parasites were enveloped in autophagosome-like vacuoles, which ultimately fused with lysosomes. These observations outline a series of mechanisms used by effector cells to redirect the fate of a classically nonfusogenic intracellular pathogen toward a path of immune elimination. JEM © The Rockefeller University Press.

The proinflammatory cytokine IL-12 drives the generation of terminally differentiated KLRG1+ effector CD8+ T cells. Using a Toxoplasma vaccination model, we delineate the sequence of events that naïve CD8+ T cells undergo to become terminal effectors and the differentiation steps controlled by IL-12. We demonstrate that direct IL-12 signaling on CD8+ T cells is essential for the induction of KLRG1 and IFN-γ, but the subsequent downregulation of CXCR3 is controlled by IL-12 indirectly through the actions of IFN-γ and IFN-γ-inducible chemokines. Differentiation of nascent effectors occurs in an extrafollicular splenic compartment and is driven by late IL-12 production by DCs distinct from the classical CD8α+ DC. Unexpectedly, we also found extensive proliferation of both KLRG1− and KLRG1+ CD8+ T cells in the marginal zone and red pulp, which ceases prior to the final KLRG1Hi CXCR3Lo stage. Our findings highlight the notion of an extrafollicular pathway for effector T cell generation. DOI: http://dx.doi.org/10.7554/eLife.09017.001, eLife digest The immune system helps to protect us from cancer, infection by microbes and other diseases. There are several different types of immune cells that each have particular roles. For example, cytotoxic T cells can kill other cells in the body that are damaged or infected. These cells are found in various locations around the body—including a region of the spleen known as the white pulp—where they wait in an inactive state until they detect signals from a damaged or infected cell. These T cells divide and mature to produce populations of active T cells known as effector cytotoxic lymphoid cells (or CTLs for short), a process which is thought to occur within the white pulp. A small protein called cytokine IL-12 is involved in the production of CTLs. The cytokine is released from other immune cells and causes the activated T cells to divide and mature. It has long been believed that IL-12 produced in the white pulp early on in the process is sufficient to drive this process, but more recent work suggests that sustained production of IL-12 in other areas of the spleen that are accessible to the bloodstream may be needed. Here, Shah et al. studied the generation of cytotoxic T cells in mice that had been exposed to a vaccine against a disease called Toxoplasmosis. Their experiments show that IL-12 drives both the early and late stages of CTL production. In the early stages, the T cells respond to IL-12 that is secreted by a group of ‘lymphoid dendritic’ cells in the white pulp. However, in the later stages, the T cells move away from the white pulp to other parts of the spleen known as the marginal zone and red pulp, where a distinct group of ‘myeloid dendritic’ cells also produce IL-12 and direct the final maturation of the CTLs. Shah et al.'s findings also show that the process in which cytotoxic T cells divide and later mature to produce CTLs involves a series of tightly controlled events that mostly occur outside of the white pulp. These observations provide a new perspective on how to develop vaccines and other treatments that more efficiently generate the CTLs needed to protect against infections and cancer. DOI: http://dx.doi.org/10.7554/eLife.09017.002

Helminth infections are ubiquitous worldwide and can trigger potent immune responses that differ from and potentially antagonize host protective responses to microbial pathogens. In this Review we focus on the three main killers in infectious disease-AIDS, tuberculosis and malaria-and critically assesses whether helminths adversely influence host control of these diseases. We also discuss emerging concepts for how M2 macrophages and helminth-modulated dendritic cells can potentially influence the protective immune response to concurrent infections. Finally, we present evidence advocating for more efforts to determine how and to what extent helminths interfere with the successful control of specific concurrent coinfections.

Schistosoma mansoni infections are associated with a strong Th2 cytokine response. Treatment of mice with IL-12 or anti-IL-2 or anti-IL-4 before i.v. injection of eggs increased IFN-gamma production and downregulated Th2 responses and pulmonary granuloma size. Conversely, anti-IFN-gamma antibody treatment increased Th2 responses and granuloma size. Similar manipulation produced less dramatic results in infected mice. However, sensitization of mice with eggs + IL-12 before infection augmented the Th1 response and decreased Th2 cytokines, granuloma size and fibrosis. Antisera to IFN-gamma, TNF-alpha or IL-12 during IL-12-egg immunization partly restored granuloma size and fibrosis following infection. Variations in the size of granulomas in acute (8 week) infections may be influenced primarily by the number and state of activation of T cells. In chronic (12-16 week) infections immunologic downmodulation proceeded normally in mice without functional CD8+ cells and in IFN-gamma KO mice but not in B cell KO (microMT) mice or in mice deficient in FcR expression in spite of the fact that these mice downregulated their T cell and cytokine responses. It is evident that the participation of cytokines in granuloma formation and regulation is complicated and that the mechanisms controlling both these phenomena are likely to involve both T cells and antibody/FcR interactions.

Various cell types in both lymphoid and non-lymphoid tissues produce the anti-inflammatory cytokine interleukin (IL)-10 during murine cytomegalovirus (MCMV) infection. The functions of IL-10 in the liver during acute infection and the cells that generate this cytokine at this site have not been extensively investigated. In this study, we demonstrate that the production of IL-10 in the liver is elevated in C57BL/6 mice during late acute MCMV infection. Using IL-10 green fluorescence protein (GFP) reporter knock-in mice, designated IL-10-internal ribosomal entry site (IRES)-GFP-enhanced reporter (tiger), NK cells are identified as major IL-10 expressing cells in the liver after infection, along with T cells and other leukocytes. In the absence of IL-10, mice exhibit marked elevations in proinflammatory cytokines and in the numbers of mononuclear cells and lymphocytes infiltrating the liver during this infection. IL-10-deficiency also enhances liver injury without improving viral clearance from this site. Collectively, the results indicate that IL-10-producing cells in the liver provide protection from collateral injury by modulating the inflammatory response associated with MCMV infection.

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2Kb-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62Lhi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses., Author Summary Toxoplasma gondii is a ubiquitous protozoan parasite that causes severe disease in people with compromised immune function. It is known that CD8+ T cells are essential for the establishment of protective immunity, primarily through the delivery of the effector cytokine interferon-γ (IFN-γ) to Toxoplasma-infected cells. However, it remains unclear how memory CD8+ T cells develop in response to Toxoplasma infection, and to what extent inflammatory cytokines like interleukin-12 (IL-12) play a role in memory development. Furthermore, the natural T. gondii antigens that induce CD8+ T cell activation have not yet been fully uncovered. Using new technology for the screening of antigen specificity, we discovered the first natural antigen-specific CD8+ T cell population induced by T. gondii infection in C57BL/6 mice. By tracking natural parasite-specific responses, we found that IL-12 plays a vital role in promoting the development of IFN-γ-producing effector memory CD8+ T cells but at a cost to the numbers and function of central memory CD8+ T cells.

CpG-DNA or its synthetic analog CpG-ODN activates innate immunity through Toll-like receptor 9 (TLR9). However, the mechanism of TLR9 activation by CpG-DNA remains elusive. Here we have identified HMGB1 as a CpG-ODN–binding protein. HMGB1 interacts and preassociates with TLR9 in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), and hastens TLR9's redistribution to early endosomes in response to CpG-ODN. CpG-ODN stimulates macrophages and dendritic cells to secrete HMGB1; in turn, extracellular HMGB1 accelerates the delivery of CpG-ODNs to its receptor, leading to a TLR9-dependent augmentation of IL-6, IL-12, and TNFα secretion. Loss of HMGB1 leads to a defect in the IL-6, IL-12, TNFα, and iNOS response to CpG-ODN. However, lack of intracellular TLR9-associated HMGB1 can be compensated by extracellular HMGB1. Thus, the DNA-binding protein HMGB1 shuttles in and out of immune cells and regulates inflammatory responses to CpG-DNA.

The granulomatous response to schistosome eggs is a CD4 T-cell-dependent, Th2-cytokine-dominated immunopathologic response. As infection proceeds to chronicity, both granuloma formation and egg-induced cytokine production become downregulated, and previous experiments have implicated CD8 T cells in this process. One mechanism by which CD8 T cells could suppress immunopathology is through the production of the counterregulatory cytokine gamma interferon (IFN-gamma), but no in vivo evidence exists to directly support this hypothesis. In this study, we analyzed hepatic granuloma formation and egg-induced cytokine production in Schistosoma mansoni-infected gene knockout mice deficient in either CD8 lymphocytes or IFN-gamma. Surprisingly, we found that neither immunologic component plays an essential function in the control of granuloma and cytokine responses during either the acute or chronic stage of infection. Thus, other mechanisms may be more important in the regulation of immunopathology in schistosomiasis.

The requirement for an architecturally intact spleen in the afferent and efferent arms of immunity to the murine malaria parasite Plasmodium chabaudi AS was analyzed. C57BL/6 mice with intact spleens develop a single, patent parasitemia and resolve the infection. In contrast, surgically splenectomized mice experience persistent waves of patent parasitemia interrupted briefly by periods of parasitologic crises. Transfer of spleen cells from immune donors, but not transfer of spleen cells from normal mice, into splenectomized mice enabled the recipients to resolve the infection similar to mice with intact spleens. B-cell depletion, but not T-cell depletion, of spleen cells prior to transfer abrogated the ability of splenectomized recipients to resolve the infection. Compared with mice with intact spleens, splenectomized mice exhibited a delayed antibody response whereas all groups of immune cell recipients had an accelerated antibody response. Nevertheless, splenectomized mice and recipients of B-cell-depleted cells failed to resolve infections, despite the development of high-titer antibodies late during the course of infection. Analysis of immunoglobulin G isotype responses showed a lower representation of immunoglobulin G2a in mice which failed to resolve infections. The latter mice had characteristic histopathologic changes in the liver. These observations indicate a unique role of the splenic microenvironment for the induction and development of an effective B-cell-dependent response against malarial parasites.

By using erythropoietin-dependent proliferation of splenic erythroid cells as an in vitro erythropoiesis model system, we demonstrate that spleen cells from Plasmodium chabaudi AS-infected C57BL/6 mice potently inhibited erythroid cell proliferation. Inhibitory activity was detected in spleen cell conditioned media (SPCM) prepared from infected mice but not from uninfected mice. The inhibitory activity in SPCM was characterized as being heat sensitive, macromolecular, and host derived. The inhibitory activity was not reversed by increasing the erythropoietin concentration and was found to be specific for the late erythroid lineage. Mouse strains, which differ in their resistance to P. chabaudi AS infection, produced and responded to the inhibitory activity to a similar extent. Putative immune mediators, interleukin 1 alpha, interleukin 1 beta, and gamma interferon, were found to be potent inhibitors of erythroid cell proliferation. However, antibody neutralization experiments failed to demonstrate a major role for these cytokines in the inhibitory activity of SPCM. Our results suggest that the elaboration of inhibitor(s) of erythropoiesis in hemopoietic organs of Plasmodium-infected mice may impair erythroid regeneration. The identity of the inhibitory mediator(s) is presently unknown but is distinct from interleukin 1, tumor necrosis factor alpha, and gamma interferon.

The Toxoplasma gondii virulence factors ROP5 and ROP18 both target immunity-related GTPases (IRGs) to evade immunity. In this issue of Cell Host & Microbe, Etheridge et al. (2014) identify a third virulence factor, ROP17, which forms a complex and synergizes with ROP5/ROP18 to fully disable the IRG system of antiparasite defense.