Effector TH17 Cells Give Rise to Long-Lived TRM Cells that Are Essential for an Immediate Response against Bacterial Infection.

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (TRM) cells. However, the cellular origin of CD4 TRM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 TRM cells derive from IL-17A-producing effector (TH17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exTH17 TRM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exTH17 TRM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 TRM cells during bacterial infection, offering novel strategies for targeted vaccine design.

Migrant memory B cells secrete luminal antibody in the vagina.

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract), dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However, owing to the paucity of polymeric immunoglobulin receptors and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead, upon secondary challenge with herpes simplex virus 2, circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-γ, which induces expression of chemokines, including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract.

Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help.

Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood-brain barrier and blood-nerve barrier, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread.

Memory Lymphocyte Clusters in Genital Immunity: Role of Tissue-Resident Memory T Cells (TRM).

Development of front-line defenses in genital tissues is important to inhibit viral/bacterial replication and to eliminate sexually transmitted diseases. In this chapter, we discuss the cellular composition, location, and function of memory lymphocyte clusters deployed in mucosal tissues and compare them with those in secondary lymphoid organs and tertiary lymphoid structures.

Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry.

Neutrophil extracellular trap (NET) formation involves the release of DNA outside the cell to neutralize pathogens. Techniques such as live microscopy, flow cytometry, and intravital imaging allow the characterization of NETs, but these either cannot be applied in vivo, lack specificity or require invasive procedures. We developed an automated analysis method to rapidly acquire and characterize cells as NETs or NET precursors, as opposed to cells undergoing other forms of cell death, using imaging flow cytometry. NETs were maintained in solution using a novel three-dimensional cell culture system in which cells are suspended at the interface of two liquids of different density. Critically, we identify NETs using an image analysis algorithm based on morphological data showing the extrusion of DNA beyond the cell boundaries. In vitro, we used this technique to demonstrate different requirements for NET formation in human and mouse neutrophils. We also measured NETs in whole blood during infection of mice with the malaria parasite Plasmodium yoelii. We expect this technique will provide a valuable approach to better understand the process of NET formation and its importance in disease. © 2019 International Society for Advancement of Cytometry.

Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice.

We previously reported that Ag85B-expressing human parainfluenza type 2 virus (Ag85B-rHPIV2) was effective as a nasal vaccine against tuberculosis in mice; however, the mechanism by which it induces an immune response remains to be investigated. In the present study, we found that organogenesis of inducible bronchus-associated lymphoid tissue (iBALT) played a role in the induction of antigen-specific T cells and IgA antibody responses in the lung of mice intra-nasally administered Ag85B-rHPIV2. We found that expression of Ag85B was dispensable for the development of iBALT, suggesting that HPIV2 acted as an iBALT-inducing vector. When iBALT organogenesis was disrupted in Ag85B-rHPIV2-immunized mice, either by neutralization of the lymphotoxin pathway or depletion of CD11b+ cells, Ag85B-specific immune responses (i.e. IFN γ-producing T cells and IgA antibody) were diminished in the lung. Furthermore, we found that immunization with Ag85B-rHPIV2 induced neutrophil and eosinophil infiltration temporally after the immunization in the lung. Thus, our results show that iBALT organogenesis contributes to the induction of antigen-specific immune responses by Ag85B-rHPIV2 and that Ag85B-rHPIV2 provokes its immune responses without inducing long-lasting inflammation.

Tissue instruction for migration and retention of TRM cells.

During infection, a subset of effector T cells seeds the lymphoid and non-lymphoid tissues and gives rise to tissue-resident memory T cells (TRM). Recent findings have provided insight into the molecular and cellular mechanisms underlying tissue instruction of TRM cell homing, as well as the programs involved in their retention and maintenance. We review these findings here, highlighting both common features and distinctions between CD4 TRM and CD8 TRM cells. In this context we examine the role of memory lymphocyte clusters (MLCs), and propose that the MLCs serve as an immediate response center consisting of TRM cells on standby, capable of detecting incoming pathogens and mounting robust local immune responses to contain and limit the spread of infectious agents.

Recruited inflammatory monocytes stimulate antiviral Th1 immunity in infected tissue.

Monocytes patrol various tissues for signs of infection and inflammation. Inflammatory monocytes enter peripheral tissues at sites of microbial infection and differentiate into dendritic cells and macrophages. Here, we examined the importance of monocytes in primary mucosal infection with herpes simplex virus 2 (HSV-2), and demonstrate that monocyte-derived APCs are required to elicit IFN-γ secretion from effector Th1 cells to mediate antiviral protection. However, monocyte-derived APCs were dispensable for the generation of Th1 immunity and for the restimulation of memory Th1 cells during secondary viral challenge. These results demonstrate that distinct APC subsets are dedicated for CD4 T cell priming, elicitation, and memory recall responses to a given viral pathogen within the same mucosal tissue and reveal a specialized role for monocyte-derived APCs in the emergency response to infection.

Increased IL-10-competent regulatory B cells in the decidua of early human pregnancy.

Regulatory B cells (Bregs) may play a pivotal role in maintaining human pregnancy. For the first time, to the best of our knowledge, this study noted that cell percentages of CD24hiCD38hi Bregs and CD24hiCD27+ Bregs, which can potentially produce IL-10, are increased in human decidua compared with the mid-luteal phase endometrium. In each case of decidua, the correlation between Bregs and dendritic cell (DC) or natural killer (NK) cell expression was further explored. A positive correlation between the percentage of CD24hiCD38hi Bregs and CD123-CD11c+ myeloid DCs (mDCs) was noted. Furthermore, a significant positive correlation was also observed between the percentage of CD24hiCD27+ Bregs and CD94+CD56brightCD16- suppressive NK cells. These findings regarding decidual Bregs deepen the understanding of the harmonious immunological microenvironment that sustains early human pregnancy.

Refeeding activates neurons in the dorsomedial hypothalamus to inhibit food intake and promote positive valence.

OBJECTIVE: The regulation of food intake is a major research area in the study of obesity, which plays a key role in the development of metabolic syndrome. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behavior, but the deletion of a gene has a long-term effect on neurophysiology. Our understanding of short-term changes such as appetite under physiological conditions is therefore still limited. METHODS: Targeted recombination in active populations (TRAP) is a newly developed method for labeling active neurons by using tamoxifen-inducible Cre recombination controlled by the promoter of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), a member of immediate early genes. Transgenic mice for TRAP were fasted overnight, re-fed with normal diet, and injected with 4-hydroxytamoxifen 1 h after the refeeding to label the active neurons. The role of labeled neurons was examined by expressing excitatory or inhibitory designer receptors exclusively activated by designer drugs (DREADDs). The labeled neurons were extracted and RNA sequencing was performed to identify genes that are specifically expressed in these neurons. RESULTS: Fasting-refeeding activated and labeled neurons in the compact part of the dorsomedial hypothalamus (DMH) that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of the labeled DMH neurons decreased food intake and developed place preference, an indicator of positive valence. Chemogenetic activation or inhibition of these neurons had no influence on the whole-body glucose metabolism. The labeled DMH neurons expressed prodynorphin (pdyn), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and thyrotropin-releasing hormone receptor (Trhr) genes. CONCLUSIONS: We identified a novel cell type of DMH neurons that can inhibit food intake and promote feeding-induced positive valence. Our study provides insight into the role of DMH and its molecular mechanism in the regulation of appetite and emotion.

Prostaglandin in the ventromedial hypothalamus regulates peripheral glucose metabolism.

The hypothalamus plays a central role in monitoring and regulating systemic glucose metabolism. The brain is enriched with phospholipids containing poly-unsaturated fatty acids, which are biologically active in physiological regulation. Here, we show that intraperitoneal glucose injection induces changes in hypothalamic distribution and amounts of phospholipids, especially arachidonic-acid-containing phospholipids, that are then metabolized to produce prostaglandins. Knockdown of cytosolic phospholipase A2 (cPLA2), a key enzyme for generating arachidonic acid from phospholipids, in the hypothalamic ventromedial nucleus (VMH), lowers insulin sensitivity in muscles during regular chow diet (RCD) feeding. Conversely, the down-regulation of glucose metabolism by high fat diet (HFD) feeding is improved by knockdown of cPLA2 in the VMH through changing hepatic insulin sensitivity and hypothalamic inflammation. Our data suggest that cPLA2-mediated hypothalamic phospholipid metabolism is critical for controlling systemic glucose metabolism during RCD, while continuous activation of the same pathway to produce prostaglandins during HFD deteriorates glucose metabolism.

Vaginal epithelial dendritic cells renew from bone marrow precursors.

Dendritic cells (DCs) represent key professional antigen-presenting cells capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by nonmonocyte bone-marrow-derived precursors, with a half-life of 13 days under steady-state conditions. Upon infection with HSV-2, the Gr-1(hi) monocytes were found to give rise to VEDCs. Furthermore, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b(+)F4/80(hi), CD11b(+)F4/80(int), and CD11b(-)F4/80(-). Importantly, these VEDC populations expressed CD207 at low levels and had a constitutively more activated phenotype compared with the skin LCs. Collectively, our results revealed mucosa-specific features of the VEDCs with respect to their phenotype, activation status, and homeostatic renewal potential.

Decidual CD68+ HLA-DR+ CD163- M1 macrophages increase in miscarriages with normal fetal chromosome.

PROBLEM: Is an abnormal increase or decrease of M1/M2 macrophages observed in the deciduae of miscarriages with normal fetal chromosome (MN)? METHODS OF STUDY: Deciduae of 18 MN and 26 miscarriages with abnormal fetal chromosome (MA) were obtained. Additionally, deciduae from 15 women whose pregnancies ended in induced abortions (IA) and endometriums at the mid-luteal phase from 19 non-pregnant women endomeriums of mid-luteal phases (EM) were obtained. Macrophages were analyzed by flow cytometry using monoclonal antibodies for CD68, HLA-DR, and CD163. RESULTS: M1 macrophages, defined as CD68+ HLA-DR+ CD163- cells, increased in MN compared with MA or IA. M2 macrophages, defined as CD68+ HLA-DR- CD163+ cells, increased in the deciduae of MA and IA compared with EM. However, this increase was not observed in the deciduae of MN. CONCLUSION: Our findings of phenotypic characters of decidual macrophages in MN provide additional evidence that M2 polarization is favorable for the maintenance of early stages of pregnancy.

Th1 or Th2 balance regulated by interaction between dendritic cells and NKT cells.

If Th1 or Th2 polarization could be artificially manipulated, effective immune responses would be generated depending on nature of the targets. In this study we attempted to regulate CD40 expressions on dendritic cells (DCs) in order to modify the T cell response. It was found that reducing agents selectively inhibited surface expression of CD40 on DCs. This finding may provide a new strategy of DC-mediated modulation of the Th1/Th2 balance. It was also shown that NKT-produced Th1/Th2 cytokine balance was under control of negative feedback loop through DCs. Th1 cytokine-pretreated DCs mainly induced Th2 cytokine production, whereas Th2 cytokine-pretreated DCs induced Th1 cytokine production by alpha-galactosylceramide-stimulated NKT cells. The negative feedback regulation system could be applicable to therapeutics of various diseases based on immunological disorders.

Divergence of helper, cytotoxic, and regulatory T cells in the decidua from miscarriage.

PROBLEM: The aim of this prospective study was to evaluate phenotypic differences of helper T (Th), cytotoxic T (Tc), and regulatory T (Treg) cells in the deciduae of missed miscarriage with a normal chromosome karyotype of a fetus (MN) and missed miscarriage with an abnormal chromosome karyotype of a fetus (MA). METHODS OF STUDY: The decidua of 19 MN and 28 MA was obtained. Additionally, the decidua of 15 induced abortion (IA) and the endometrium of 19 non-pregnant women (EM) were obtained. IFN-γ(+) , IL-17(+) , CD25(high) Foxp3(+) cells in CD4(+) (Th) cells, and IFN-γ(+) cells in CD8(+) (Tc) cells were evaluated by flow cytometry. RESULTS: The percentages of IFN-γ(+) Tc and CD4(+) CD25(high) Foxp3(+) (Treg) cells in MN were significantly increased as compared with MA and IA. The percentage of IFN-γ(+) Th in MN was increased as compared with IA. CONCLUSION: Activation of IFN-γ(+) Tc and Treg cells in the decidua might be associated with the pathophysiology underlying MN.

Activation of extracellular signal-related kinase by TNF-alpha controls the maturation and function of murine dendritic cells.

Functional roles of extracellular signal-related kinase (ERK) activation in dendritic-cell (DC) maturation have been unclear. In the present study, we investigated the ERK pathway in tumor necrosis factor (TNF)-alpha-induced maturation of murine spleen-derived DC. TNF-alpha increased surface expressions of major histocompatibility (MHC) and costimulatory molecules on DC in a dose-dependent manner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF-alpha markedly enhanced ERK1/2 activation in DC, and this activation was blocked completely by PD98059, a selective inhibitor of the ERK pathway. When DC were treated with TNF-alpha at a low but not a high concentration, PD98059 notably enhanced surface expressions of the MHC and costimulatory molecules and allostimulatory capability of the DC. Interleukin (IL)-12 production was enhanced significantly by PD98059 in DC treated with low or high concentration of TNF-alpha. These findings suggest that TNF-alpha-induced ERK activation negatively controls maturation and IL-12 production in murine DC.

T cell memory. A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells.

CD8 tissue-resident memory T (T(RM)) cells provide efficient local control of viral infection, but the role of CD4 T(RM) is less clear. Here, by using parabiotic mice, we show that a preexisting pool of CD4 T(RM) cells in the genital mucosa was required for full protection from a lethal herpes simplex virus 2 (HSV-2) infection. Chemokines secreted by a local network of macrophages maintained vaginal CD4 T(RM) in memory lymphocyte clusters (MLCs), independently of circulating memory T cells. CD4 T(RM) cells within the MLCs were enriched in clones that expanded in response to HSV-2. Our results highlight the need for vaccine strategies that enable establishment of T(RM) cells for protection from a sexually transmitted virus and provide insights as to how such a pool might be established.

High-risk human papillomavirus E6 inhibits monocyte differentiation to Langerhans cells.

High-risk human papillomaviruses (HPVs) cause a variety of malignancies of the mucosal epithelium. However, the local immune evasion strategies used by HPV-transformed cells remain unclear. Here, we examined the effect of HPV-positive cancer cells on human peripheral blood monocytes, which are precursors of Langerhans cells, key antigen-presenting cells in the squamous epithelium. HPV-positive cervical cancer cells and HPV-E6 expressing cells inhibited monocyte differentiation to Langerhans cells in a contact-dependent manner. Unlike Langerhans cells, monocytes that differentiated in the presence of HPV16 E6-expressing cells exhibited high levels of endocytic activity. Our results suggest that cells infected by high-risk HPV evade immune surveillance by blocking the differentiation of monocytes into competent antigen presenting cells.

A neuron-specific role for autophagy in antiviral defense against herpes simplex virus.

Type I interferons (IFNs) are considered to be the universal mechanism by which viral infections are controlled. However, many IFN-stimulated genes (ISGs) rely on antiviral pathways that are toxic to host cells, which may be detrimental in nonrenewable cell types, such as neurons. We show that dorsal root ganglionic (DRG) neurons produced little type I IFNs in response to infection with a neurotropic virus, herpes simplex type 1 (HSV-1). Further, type I IFN treatment failed to completely block HSV-1 replication or to induce IFN-primed cell death in neurons. We found that DRG neurons required autophagy to limit HSV-1 replication both in vivo and in vitro. In contrast, mucosal epithelial cells and other mitotic cells responded robustly to type I IFNs and did not require autophagy to control viral replication. These findings reveal a fundamental difference in the innate antiviral strategies employed by neurons and mitotic cells to control HSV-1 infection.