Peter Velazquez

Monocyte trafficking to hepatic sites of bacterial infection is chemokine independent and directed by focal intercellular adhesion molecule-1 expression.

Recruitment of CCR2+Ly6Chigh monocytes to sites of infection is essential for efficient clearance of microbial pathogens. Although CCR2-mediated signals promote monocyte emigration from bone marrow, the contribution of CCR2 to later stages of monocyte recruitment remains unresolved. In this article, we show that CCR2 deficiency markedly worsens hepatic Listeria monocytogenes infection because Ly6Chigh monocytes are retained in the bone marrow. Intravenously transferred, CCR2-deficient Ly6Chigh monocytes traffic normally to hepatic foci of infection and contribute to bacterial clearance. Pertussis toxin treatment of adoptively transferred monocytes does not impair their intrahepatic trafficking, suggesting that chemokine signaling, once CCR2+Ly6Chigh monocytes emigrate from the bone marrow, is not required for monocyte localization to sites of bacterial infection in the liver. Expression of ICAM-1 is induced in close proximity to foci of bacterial infection in the liver, including on CD31+ endothelial cells, and blockade of CD11b and CD44 diminishes monocyte localization to these hepatic foci. Our studies demonstrated that Ly6Chigh monocyte recruitment from the bloodstream to the L. monocytogenes-infected liver does not require chemokine receptor-mediated signals but instead is principally dependent on integrin- and extracellular matrix-mediated monocyte adhesion.

Cutting Edge: Activation by Innate Cytokines or Microbial Antigens Can Cause Arrest of Natural Killer T Cell Patrolling of Liver Sinusoids

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of α-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-γ, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-γ production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, α-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.

Formation of B and T cell subsets require the cannabinoid receptor CB2

A recent and surprising body of research has linked changes in immune function to biologic and therapeutic targeting of cannabinoid receptors, which prototypically respond to delta-9 tetrahydrocannabinol. The peripheral cannabinoid receptor CB2 is highly expressed in immune cell types (macrophages, dendritic cells, and B cells), and pharmacologically alters their cytokine production and responsiveness. Accordingly, cannabinoid agonists can powerfully alter susceptibility to certain microbial infections, atherosclerosis, and cancer immunotherapy. What is unknown is the physiologic role of natural levels of endocannabinoids and their receptors in normal immune homeostasis. Gαi2−/− mice are deficient in the formation of certain B and T cell subsets and are susceptible to immune dysregulation, notably developing inflammatory bowel disease. A key issue is the identity of the Gi-coupled receptors relevant to this Gαi2-signaling pathway. We find that mice deficient in CB2, the Gi-coupled peripheral endocannabinoid receptor, have profound deficiencies in splenic marginal zone, peritoneal B1a cells, splenic memory CD4+ T cells, and intestinal natural killer cells and natural killer T cells. These findings partially phenocopy and extend the lymphocyte developmental disorder associated with the Gαi2−/− genotype, and suggest that the endocannabinoid system is required for the formation of T and B cell subsets involved in immune homeostasis. This noncompensatable requirement for physiologic function of the endocannabinoid system is novel. Because levels of endocannabinoids are highly restricted microanatomically, local regulation of their production and receptor expression offers a new principle for regional immune homeostasis and disease susceptibility, and extends and refines the rationale for CB2-targeted immunotherapy in immune and inflammatory diseases.

Surveillance B lymphocytes and mucosal immunoregulation.

Mucosal lymphocyte homeostasis involves the dynamic interaction of enteric microbiota, the intestinal host epithelium, and the mucosal immune system. Dysregulation of mucosal lymphocyte homeostasis results in a variety of intestinal disorders, notably inflammatory bowel diseases like ulcerative colitis and Crohn’s disease. One key cellular component regulating homeostasis are B lymphocytes that reside in gut-associated lymphoid tissue. This compartment includes Peyer’s patches, isolated lymphoid follicles, lamina propria, and mesenteric lymph nodes. Recent data have pointed to two new and exciting aspects of B cells in the gut. First, there has been progress on identification and functional analysis of abundant isolated lymphoid follicle B cells that are key mediators of IgA genesis. Second, several groups have now clarified the functional identification and characterization of immunoregulatory B cells in the gut. This review examines the novel aspects of these B cells, and examines how each plays a role in mediating mucosal homeostasis in this bacteria-laden compartment.

Villous B Cells of the Small Intestine Are Specialized for Invariant NK T Cell Dependence

B cells are important in mucosal microbial homeostasis through their well-known role in secretory IgA production and their emerging role in mucosal immunoregulation. Several specialized intraintestinal B cell compartments have been characterized, but the nature of conventional B cells in the lamina propria is poorly understood. In this study, we identify a B cell population predominantly composed of surface IgM+ IgD+ cells residing in villi of the small intestine and superficial lamina propria of the large intestine, but distinct from the intraepithelial compartment or organized intestinal lymphoid structures. Small intestinal (villous) B cells are diminished in genotypes that alter the strength of BCR signaling (Bruton tyrosine kinasexid, Gαi2−/−), and in mice lacking cognate BCR specificity. They are not dependent on enteric microbial sensing, because they are abundant in mice that are germfree or genetically deficient in TLR signaling. However, villous B cells are reduced in the absence of invariant NK T cells (Jα18−/− or CD1d−/− mice). These findings define a distinct population of conventional B cells in small intestinal villi, and suggest an immunologic link between CD1-restricted invariant NK T cells and this B cell population.

Integration of B cells and CD8 + T in the protective regulation of systemic epithelial inflammation

Mechanisms that control abnormal CD4(+) T cell-mediated tissue damage are a significant factor in averting and resolving chronic inflammatory epithelial diseases. B cells can promote such immunoregulation, and this is thought to involve interaction with MHC II- or CD1-restricted regulatory T cells. The purpose of this study is to genetically define the interacting cells targeted by protective B cells, and to elucidate their regulatory mechanisms in CD4(+) T cell inflammation. Transfer of G alpha i2-/- CD3(+) T cells into lymphopenic mice causes a dose-dependent multi-organ inflammatory disease including the skin, intestine, and lungs. Disease activity is associated with elevated levels of serum TNF-alpha and IFN-gamma, and an activated IL-17 producing CD4(+) T cell population. Mesenteric node B cells from wild type mice suppress disease activity, serum cytokine expression, and levels of CD4(+) T cells producing TNF-alpha IFN-gamma, and IL-17. The protective function of B cells requires genetic sufficiency of IL-10, MHC I and TAP1. Regulatory B cells induce the expansion and activation of CD8(+) T cells, which is correlated with disease protection. These results demonstrate that CD8(+) T cells can ameliorate lymphopenic systemic inflammatory disease, through peptide/MHC I-dependent B cell interaction.

Visualization of mucosal homeostasis via single‐ and multiphoton intravital fluorescence microscopy

FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single‐ and multiphoton FIVM. Tissue damage is limited to ∼200 μm, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran‐FITC and 70 KDa dextran‐TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL‐17F locus were used to track DC subsets, FoxP3+ Tregs, or Th17f cells, respectively. Resident cx3cr1+/egfp cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1+ DCs and T cells display activity similar to that found in steady‐state, secondary lymphoid organs.

Renal dendritic cells: an update.

Discovery into the role of renal dendritic cells (rDCs) in health and disease of the kidney is rapidly accelerating. Progress in deciphering DC precursors and the heterogeneity of monocyte subsets in mice and humans is providing insight into the biology of rDCs. Recent findings have extended knowledge of the origins, anatomy and function of the rDC network at steady state and during periods of injury to the renal parenchyma. This brief review highlights these new findings and provides an update on the study of rDCs.

Dynamics of host defense: the view at the front lines

Relatively few studies have used live tissue microscopy to evaluate how the immune system responds to pathogens. In this commentary we discuss the challenges of imaging infectious processes and the questions that can be addressed with these dynamic approaches.

Distinct behavior of myelomonocytic cells and CD8 T cells underlies the hepatic response to Listeria monocytogenes

Background: The immune response to Listeria monocytogenes (LM) is characterized by formation of leukocyte rich foci of infection in liver and spleen. Although much has been gained in our understanding of immune response through the study of LM, little is known about spatio-temporal regulation of immune response to Listeria in liver. Methods: We utilize a combination of molecular, genetic and intravital microscopic approaches to gain insight into the dynamics of foci and leukocyte behavior during hepatic Listeriosis. Results: LM foci efficiently exclude blood flow, indicating the presence of a barrier separating the foci and healthy tissue. Despite this barrier, sinusoidal myelomonocytic cells readily enter or transiently interact with cells at the edge of foci of infection. Next, utilizing L9.6 transgenic CD8 + T cells specific for an endogenously processed LM antigen, p60 217-225, along with LM deficient in this epitope, we define the role of TCR in T cell migratory behavior in infected liver. Surprisingly, T cell behavior varies with micro-anatomic locale. Near foci, non-specific adhesion mechanisms dominate lymphocyte behavior. Antigen specific effects on motility became detectable only distal to foci. Conclusions: These data suggest that LM antigens act in a paracrine manner to mediate protection from Listeriosis in the liver.