A Functional Assessment of Fetal Liver and Monocyte-Derived Macrophages in the Lung Alveolar Environment.

It is becoming clear that every organ is seeded by a population of fetal liver-derived macrophages that are replaced at different rates by monocyte-derived macrophages. Using the Ms4a3tdTomato reporter mouse that reports on monocyte-derived alveolar macrophages (Mo-AMs) and our ability to examine AM function using our multichannel intravital microscopy, we examined the fetal-liver derived alveolar macrophage (FL-AM) and Mo-AM populations within the same mouse under various environmental conditions. The experiments unveiled that AMs migrated from alveolus to alveolus and phagocytosed bacteria identically regardless of ontogenic origin. Using 50 PFU of influenza A virus (IAV) determined using the Madin-Darby canine kidney (MDCK) cell line, we noted that both populations were susceptible to IAV-induced immunoparalysis, which also led to impaired phagocytosis of secondary bacterial infections. Both FL-AMs and Mo-AMs were trained by ß-glucan to resist IAV-induced paralysis. Over time (40 wk), Mo-AMs began to outperform FL-AMs, although both populations were still sensitive to IAV. Our data also show that clodronate depletion of AMs leads to replenishment, but by FL-AMs, and these macrophages do show some functional impairment for a limited time. Overall, the system is designed such that new macrophages rapidly assume the function of tissue-resident macrophages when both populations are examined in an identical environment. These data do differ from artificial depletion methods that compare Mo-AMs and FL-AMs.

Imaging reveals novel innate immune responses in lung, liver, and beyond.

Highly dynamic immune responses are generated toward pathogens or injuries, in vivo. Multiple immune cell types participate in various facets of the response which leads to a concerted effort in the removal and clearance of pathogens or injured tissue and a return to homeostasis. Intravital microscopy (IVM) has been extensively utilized to unravel the dynamics of immune responses, visualizing immune cell behavior in intact living tissues, within a living organism. For instance, the phenomenon of leukocyte recruitment cascade. Importantly, IVM has led to a deep appreciation that immune cell behavior and responses in individual organs are distinct, but also can influence one another. In this review, we discuss how IVM as a tool has been used to study the innate immune responses in various tissues during homeostasis, injury, and infection.

Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis.

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Invariant natural killer T-cell development and function with loss of microRNA-155.

Invariant natural killer T (iNKT) cells are adaptive T cells with innate-like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR-155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon-γ expression. We examined the role of miR-155 in the development and function of iNKT cells. Using germline-deficient miR-155 mice, we showed that loss of miR-155 resulted in unchanged iNKT cell frequency and cell number. Although miR-155 was up-regulated in iNKT cells upon activation with α-galactosylceramide, loss of miR-155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR-155 expression.

Bispecific antibody targets multiple Pseudomonas aeruginosa evasion mechanisms in the lung vasculature.

Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an anti-Psl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa.

iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation.

iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

Expression of NALPs in adipose and the fibrotic progression of non-alcoholic fatty liver disease in obese subjects.

BACKGROUND: Visceral obesity is often accompanied by non-alcoholic fatty liver disease (NAFLD). Activation of NACHT, LRR and PYD domains-containing proteins (NALPs) may contribute to the release of pro-inflammatory cytokines by adipose and the obesity-associated progression of NAFLD to non-alcoholic steatohepatitis (NASH). METHODS: We analyzed visceral adipose expression of various NALPs and its downstream effectors caspase-1, ASC (Apoptosis-associated speck-like protein containing a CARD), IL-18 (Interleukin-18) and IL-1ß (Interleukin- 1Beta) in obese subjects (BMI ≥ 35) with biopsy proven NAFLD. RESULTS: In adipose samples collected from NASH and pericellular fibrosis patients cohorts, expression levels of NALPs and IL-1ß were lower than that in non-NASH patients. In portal fibrosis, the levels of mRNA encoding anti-inflammatory NALP6 were upregulated. The expression levels of all NALPs were significantly co-correlated. Circulating IL-18 levels were associated with increased liver injury markers AST and ALT and portal fibrosis. CONCLUSION: Our observations point at a possible shift in inflammation and fibrotic response from adipose tissue to liver and a possible negative feedback regulation of tissue inflammation that may instigate NAFLD severity.

Expression of inflammation-related genes is altered in gastric tissue of patients with advanced stages of NAFLD.

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.