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Shigella infection is the second leading cause of death due to diarrheal disease in young children worldwide. With the rise of antibiotic resistance, initiatives to design and deploy a safe and effective Shigella vaccine are urgently needed. However, efforts to date have been hindered by the limited understanding of immunological correlates of protection against shigellosis. We applied systems serology to perform a comprehensive analysis of Shigella-specific antibody responses in sera obtained from volunteers before and after experimental infection with S. flexneri 2a in a series of controlled human challenge studies. Polysaccharide-specific antibody responses are infrequent prior to infection and evolve concomitantly with disease severity. In contrast, pre-existing antibody responses to type 3 secretion system proteins, particularly IpaB, consistently associate with clinical protection from disease. Linked to particular Fc-receptor binding patterns, IpaB-specific antibodies leverage neutrophils and monocytes, and complement and strongly associate with protective immunity. IpaB antibody-mediated functions improve with a subsequent rechallenge resulting in complete clinical protection. Collectively, our systems serological analyses indicate protein-specific functional correlates of immunity against Shigella in humans.

Pathogen-specific immunity evolves in the context of the infected tissue. However, current immune correlates analyses and vaccine efficacy metrics are based on immune functions from peripheral cells. Less is known about tissue-resident mechanisms of immunity. While antibodies represent the primary correlate of immunity following most clinically approved vaccines, how antibodies interact with localized, compartment-specific immune functions to fight infections, remains unclear. Emerging data demonstrate a unique community of immune cells that reside within different tissues. These tissue-specific immunological communities enable antibodies to direct both expected and unexpected local attack strategies to control, disrupt, and eliminate infection in a tissue-specific manner. Defining the full breadth of antibody effector functions, how they selectively contribute to control at the site of infection may provide clues for the design of next-generation vaccines able to direct the control, elimination, and prevention of compartment specific diseases of both infectious and non-infectious etiologies.

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.

Human cytomegalovirus (HCMV) causes a lifelong infection through establishment of latency. Although reactivation from latency can cause life-threatening disease, our molecular understanding of HCMV latency is incomplete. Here we use single cell RNA-seq analysis to characterize latency in monocytes and hematopoietic stem and progenitor cells (HSPCs). In monocytes, we identify host cell surface markers that enable enrichment of latent cells harboring higher viral transcript levels, which can reactivate more efficiently, and are characterized by reduced intrinsic immune response that is important for viral gene expression. Significantly, in latent HSPCs, viral transcripts could be detected only in monocyte progenitors and were also associated with reduced immune-response. Overall, our work indicates that regardless of the developmental stage in which HCMV infects, HCMV drives hematopoietic cells towards a weaker immune-responsive monocyte state and that this anergic-like state is crucial for the virus ability to express its transcripts and to eventually reactivate., eLife digest Most people around the world unknowingly carry the human cytomegalovirus, as this virus can become dormant after infection and hide in small numbers of blood stem cells (which give rise to blood and immune cells). Dormant viruses still make their host cells read their genetic information and create viral proteins – a process known as gene expression – but they do not use them to quickly multiply. However, it is possible for the cytomegalovirus to reawaken at a later stage and start replicating again, which can be fatal for people with weakened immune systems. It is therefore important to understand exactly how the virus can stay dormant, and how it reactivates. Only certain infected cells allow dormant viruses to later reactivate; in others, it never starts to multiply again. Techniques that can monitor individual cells are therefore needed to understand how the host cells and the viruses interact during dormant infection and reactivation. To investigate this, Shnayder et al. infected blood stem cells in the laboratory and used a method known as single-cell RNA analysis, which highlights all the genes (including viral genes) that are expressed in a cell. This showed that in certain cells, the virus dampens the cell defenses, leading to a higher rate of viral gene expression and, in turn, easier reactivation. Further experiments showed that the blood stem cells that expressed the viral genes were marked to become a type of immune cells known as monocytes. In turn, these infected monocytes were shown to be less able to defend the body against infection, suggesting that latent human cytomegalovirus suppresses the body’s innate immune response. The reactivation of human cytomegalovirus is a dangerous issue for patients who have just received an organ or blood stem cells transplant. The study by Shnayder et al. indicates that treatments that boost innate immunity may help to prevent the virus from reawakening, but more work is needed to test this theory.

Summary Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer., Graphical Abstract, Highlights • ASL CKO enables dissection of NO contribution to disease in a cell-specific manner • NO has different roles in the specific cell types relevant to colitis • NO synthesis in enterocytes is a native defense mechanism against colitis • Metabolic modulation of NO levels is beneficial for colitis and associated colon cancer, ASL levels metabolically regulate NO synthesis in a cell-specific manner. Here, we find that cell-autonomous production of NO by enterocytes can be protective as part of the innate immune response against colitis. Finally, we demonstrate the superior advantage of metabolic modulation as a therapy for colitis and inflammation-associated colon cancer.

Monocytes are circulating short-lived macrophage precursors that are recruited on demand from the blood to sites of inflammation and challenge. In steady state, classical monocytes give rise to vasculature-resident cells that patrol the luminal side of the endothelium. In addition, classical monocytes feed macrophage compartments of selected organs, including barrier tissues, such as the skin and intestine, as well as the heart. Monocyte differentiation under conditions of inflammation has been studied in considerable detail. In contrast, monocyte differentiation under non-inflammatory conditions remains less well understood. Here we took advantage of a combination of cell ablation and precursor engraftment to investigate the generation of gut macrophages from monocytes. Collectively, we identify factors associated with the gradual adaptation of monocytes to tissue residency. Moreover, comparison of monocyte differentiation into the colon and ileum-resident macrophages revealed the graduated acquisition of gut segment-specific gene expression signatures.

Several cytokines maintain intestinal health, but precise inter-cellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue, critical for gut homeostasis. Here we show that in a murine IBD model based on macrophage-restricted Interleukin 10 (IL-10) receptor deficiency (Cx3cr1(Cre):Il10ra(fl/fl) mice), pro-inflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition of children carrying IL10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1(Cre): Il10ra(fl/fl):Il23a(fl/fl) mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to pro-inflammatory macrophages, we provide evidence that T cells of colitic animals produce deleterious IL-22 that induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define critical cell-type-specific contributions to the induction and effector mechanism of macrophage-driven colitis, as manifested in mice harboring IL-10R deficiencies and human IBD pathologies.

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study.

Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll‐like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2‐p) ameliorated DSS‐induced colitis by interfering specifically with the activation of Ly6C + monocytes without affecting their recruitment to the colon. We report that TLR2‐p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal‐regulated kinases (ERK) signaling and reduced secretion of pro‐inflammatory cytokines, such as interleukin (IL)‐6, IL‐23, IL‐12, and IL‐1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro‐inflammatory Ly6C hi monocytes and suggests that inhibition of this aggregation by TLR2‐p might have therapeutic potential in the treatment of acute gut inflammation.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels. Light-induced norepinephrine and TNF secretion augments HSPC differentiation and increases vascular permeability to replenish the blood. In contrast, darkness-induced TNF increases melatonin secretion to drive renewal of HSPCs and LT-HSC potential through modulating surface CD150 and c-Kit expression, increasing COX-2/αSMA+ macrophages, diminishing vascular permeability, and reducing HSPC ROS levels. These findings reveal that light- and darkness-induced daily bursts of norepinephrine, TNF, and melatonin within the BM are essential for synchronized mature blood cell production and HSPC pool repopulation.

SummaryInterleukin-10 (IL-10) is a pleiotropic anti-inflammatory cytokine produced and sensed by most hematopoietic cells. Genome-wide association studies and experimental animal models point at a central role of the IL-10 axis in inflammatory bowel diseases. Here we investigated the importance of intestinal macrophage production of IL-10 and their IL-10 exposure, as well as the existence of an IL-10-based autocrine regulatory loop in the gut. Specifically, we generated mice harboring IL-10 or IL-10 receptor (IL-10Rα) mutations in intestinal lamina propria-resident chemokine receptor CX3CR1-expressing macrophages. We found macrophage-derived IL-10 dispensable for gut homeostasis and maintenance of colonic T regulatory cells. In contrast, loss of IL-10 receptor expression impaired the critical conditioning of these monocyte-derived macrophages and resulted in spontaneous development of severe colitis. Collectively, our results highlight IL-10 as a critical homeostatic macrophage-conditioning agent in the colon and define intestinal CX3CR1hi macrophages as a decisive factor that determines gut health or inflammation.

The study of the intestinal dendritic cell (DC) compartment, its homeostasis, regulation, and response to challenges calls for the investigation within the physiological tissue context comprising the unique anatomic constellation of the epithelial single cell layer and the luminal microbiota, as well as neighboring immune and nonimmune cells. Here we provide protocols we developed that use a combination of conditional cell ablation, conditional compartment mutagenesis, and adoptive precursor transfers to study DC and other intestinal mononuclear phagocytes in in vivo context. We will highlight pitfalls and strengths of these approaches.

The study of the intestinal dendritic cell (DC) compartment, its homeostasis, regulation, and response to challenges calls for the investigation within the physiological tissue context comprising the unique anatomic constellation of the epithelial single cell layer and the luminal microbiota, as well as neighboring immune and nonimmune cells. Here we provide protocols we developed that use a combination of conditional cell ablation, conditional compartment mutagenesis, and adoptive precursor transfers to study DC and other intestinal mononuclear phagocytes in in vivo context. We will highlight pitfalls and strengths of these approaches.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.

Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.