Long-Term Programming of CD8 T Cell Immunity by Perinatal Exposure to Glucocorticoids.

Early life environmental exposure, particularly during perinatal period, can have a life-long impact on organismal development and physiology. The biological rationale for this phenomenon is to promote physiological adaptations to the anticipated environment based on early life experience. However, perinatal exposure to adverse environments can also be associated with adult-onset disorders. Multiple environmental stressors induce glucocorticoids, which prompted us to investigate their role in developmental programming. Here, we report that perinatal glucocorticoid exposure had long-term consequences and resulted in diminished CD8 T cell response in adulthood and impaired control of tumor growth and bacterial infection. We found that perinatal glucocorticoid exposure resulted in persistent alteration of the hypothalamic-pituitary-adrenal (HPA) axis. Consequently, the level of the hormone in adults was significantly reduced, resulting in decreased CD8 T cell function. Our study thus demonstrates that perinatal stress can have long-term consequences on CD8 T cell immunity by altering HPA axis activity.

Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function.

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates many functions of these organelles that allow phagosomes to participate in processes that are essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3 inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3 inflammasome and caspase-1 in host defense.

Pathogen-derived effectors trigger protective immunity via activation of the Rac2 enzyme and the IMD or Rip kinase signaling pathway.

Although infections with virulent pathogens often induce a strong inflammatory reaction, what drives the increased immune response to pathogens compared to nonpathogenic microbes is poorly understood. One possibility is that the immune system senses the level of threat from a microorganism and augments the response accordingly. Here, focusing on cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli-derived effector molecule, we showed the host indirectly sensed the pathogen by monitoring for the effector that modified RhoGTPases. CNF1 modified Rac2, which then interacted with the innate immune adaptors IMD and Rip1-Rip2 in flies and mammalian cells, respectively, to drive an immune response. This response was protective and increased the ability of the host to restrict pathogen growth, thus defining a mechanism of effector-triggered immunity that contributes to how metazoans defend against microbes with pathogenic potential.

Lactic acid induces transcriptional repression of macrophage inflammatory response via histone acetylation.

Lactic acid has emerged as an important modulator of immune cell function. It can be produced by both gut microbiota and the host metabolism at homeostasis and during disease states. The production of lactic acid in the gut microenvironment is vital for tissue homeostasis. In the present study, we examined how lactic acid integrates cellular metabolism to shape the epigenome of macrophages during pro-inflammatory response. We found that lactic acid serves as a primary fuel source to promote histone H3K27 acetylation, which allows the expression of immunosuppressive gene program including Nr4a1. Consequently, macrophage pro-inflammatory function was transcriptionally repressed. Furthermore, the histone acetylation induced by lactic acid promotes a form of long-term immunosuppression ("trained immunosuppression"). Pre-exposure to lactic acid induces lipopolysaccharide tolerance. These findings thus indicate that lactic acid sensing and its effect on chromatin remodeling in macrophages represent a key homeostatic mechanism that can provide a tolerogenic tissue microenvironment.

Single cell atlas of human gastric muscle immune cells and macrophage-driven changes in idiopathic gastroparesis.

Gastrointestinal immune cells, particularly muscularis macrophages (MM) interact with the enteric nervous system and influence gastrointestinal motility. Here we determine the human gastric muscle immunome and its changes in patients with idiopathic gastroparesis (IG). Single cell sequencing was performed on 26,000 CD45+ cells obtained from the gastric tissue of 20 subjects. We demonstrate 11 immune cell clusters with T cells being most abundant followed by myeloid cells. The proportions of cells belonging to the 11 clusters were similar between IG and controls. However, 9/11 clusters showed 578-11,429 differentially expressed genes. In IG, MM had decreased expression of tissue-protective and microglial genes and increased the expression of monocyte trafficking and stromal activating genes. Furthermore, in IG, IL12 mediated JAK-STAT signaling involved in the activation of tissue-resident macrophages and Eph-ephrin signaling involved in monocyte chemotaxis were upregulated. Patients with IG had a greater abundance of monocyte-like cells. These data further link immune dysregulation to the pathophysiology of gastroparesis.

Mannose-binding lectin and innate immunity.

Innate immunity is the earliest response to invading microbes and acts to contain infection in the first minutes to hours of challenge. Unlike adaptive immunity that relies upon clonal expansion of cells that emerge days after antigenic challenge, the innate immune response is immediate. Soluble mediators, including complement components and the mannose binding lectin (MBL) make an important contribution to innate immune protection and work along with epithelial barriers, cellular defenses such as phagocytosis, and pattern-recognition receptors that trigger pro-inflammatory signaling cascades. These four aspects of the innate immune system act in concert to protect from pathogen invasion. Our work has focused on understanding the protection provided by this complex defense system and, as discussed in this review, the particular contribution of soluble mediators such as MBL and phagocytic cells. Over the past two decades both human epidemiological data and mouse models have indicated that MBL plays a critical role in innate immune protection against a number of pathogens. As demonstrated by our recent in vitro work, we show that MBL and the innate immune signaling triggered by the canonical pattern-recognition receptors (PRRs), the Toll-like receptors (TLRs), are linked by their spatial localization to the phagosome. These observations demonstrated a novel role for MBL as a TLR co-receptor and establishes a new paradigm for the role of opsonins, which we propose to function not only to increase microbial uptake but also to spatially coordinate, amplify, and synchronize innate immune defenses mechanism. In this review we discuss both the attributes of MBL that make it a unique soluble pattern recognition molecule and also highlight its broader role in coordinating innate immune activation.

Blockade of CCR1 induces a phenotypic shift in macrophages and triggers a favorable antilymphoma activity.

Tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) play an important role in tumor growth and progression. TAMs have been involved in producing immunosuppressive TME via various factors; however, the underlying mechanisms remain unclear in B-cell lymphoma, including mantle cell lymphoma (MCL). We identified that chemokine receptor-1 (CCR1) is highly expressed on monocytes (Mo) and macrophages (MΦ), and CCR1 pharmacological inhibition or CCR1 siRNA abolished lymphoma-mediated Mo/MΦ migration in a chemotaxis assay. The deficiency of host CCR1 (CCR1 KO) was associated with decreased infiltration of peritoneal-MΦ compared with WT-CCR1. Functional studies indicated that the genetic depletion of CCR1 or treatment inhibited protumor MΦ (M2-like) phenotype by decreasing CD206 and IL-10 expression. Moreover, CCR1 depletion reprogrammed MΦ toward an MHCII+/TNFα+ immunogenic phenotype. Mechanistically, protumor MΦ driven-IL-10 provides a positive feedback loop to tumor-CCL3 by regulating the CCL3 promoter via STAT1 signaling. Therapeutic in vivo targeting of CCR1 with CCR1 antagonist BX-471 significantly reduced FC-muMCL1 mouse tumors in the syngeneic MCL model by the depletion of M2-TAMs and increased infiltration of cytotoxic CD8+ T cells. Our study established that CCR1 exerts a pivotal role in macrophage programming, thus shaping protumor TME and lymphoma progression. CCR1 inhibition through CCR1 antagonists may be a promising therapeutic strategy to reprogram macrophages in lymphoma-TME and achieve better clinical outcomes in patients.

Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus.

Innate immunity is vital for protection from microbes and is mediated by humoral effectors, such as cytokines, and cellular immune defenses, including phagocytic cells (e.g., macrophages). After internalization by phagocytes, microbes are delivered into a phagosome, a complex intracellular organelle with a well-established and important role in microbial killing. However, the role of this organelle in cytokine responses and microbial sensing is less well defined. In this study, we assess the role of the phagosome in innate immune sensing and demonstrate the critical interdependence of phagocytosis and pattern recognition receptor signaling during response to the Gram-positive bacteria Staphylococcus aureus. We show that phagocytosis is essential to initiate an optimal MyD88-dependent response to Staphylococcus aureus. Prior to TLR-dependent cytokine production, bacteria must be engulfed and delivered into acidic phagosomes where acid-activated host enzymes digest the internalized bacteria to liberate otherwise cryptic bacterial-derived ligands that initiate responses from the vacuole. Importantly, in macrophages in which phagosome acidification is perturbed, the impaired response to S. aureus can be rescued by the addition of lysostaphin, a bacterial endopeptidase active at neutral pH that can substitute for the acid-activated host enzymes. Together, these observations delineate the interdependence of phagocytosis with pattern recognition receptor signaling and suggest that therapeutics to augment functions and signaling from the vacuole may be useful strategies to increase host responses to S. aureus.

Low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection.

BACKGROUND: Previous studies have shown associations between low mannose-binding lectin (MBL) level or variant MBL2 genotype and sepsis susceptibility. However, MBL deficiency has not been rigorously defined, and associations with sepsis outcomes have not been subjected to multivariable analysis. METHODS: We reanalyzed MBL results in a large cohort with use of individual data from 4 studies involving a total of 1642 healthy control subjects and systematically defined a reliable deficiency cutoff. Subsequently, data were reassessed to extend previous MBL and sepsis associations, with adjustment for known outcome predictors. We reanalyzed individual data from 675 patients from 5 adult studies and 1 pediatric study of MBL and severe bacterial infection. RESULTS: XA/O and O/O MBL2 genotypes had the lowest median MBL concentrations. Receiver operating characteristic analysis revealed that an MBL cutoff value of 0.5 microg/mL was a reliable predictor of low-producing MBL2 genotypes (sensitivity, 82%; specificity, 82%; negative predictive value, 98%). MBL deficiency was associated with increased likelihood of death among patients with severe bacterial infection (odds ratio, 2.11; 95% confidence interval, 1.30-3.43). In intensive care unit-based studies, there was a trend toward increased risk of death among MBL-deficient patients (odds ratio, 1.58; 95% confidence interval, 0.90-2.77) after adjustment for Acute Physiology and Chronic Health Enquiry II score. The risk of death was increased among MBL-deficient patients with Streptococcus pneumoniae infection (odds ratio, 5.62; 95% confidence interval, 1.27-24.92) after adjustment for bacteremia, comorbidities, and age. CONCLUSIONS: We defined a serum level for MBL deficiency that can be used with confidence in future studies of MBL disease associations. The risk of death was increased among MBL-deficient patients with severe pneumococcal infection, highlighting the pathogenic significance of this innate immune defence protein.

Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages.

Interleukin 10 (IL-10) is an anti-inflammatory cytokine that plays a critical role in the control of immune responses. However, its mechanisms of action remain poorly understood. Here, we show that IL-10 opposes the switch to the metabolic program induced by inflammatory stimuli in macrophages. Specifically, we show that IL-10 inhibits lipopolysaccharide-induced glucose uptake and glycolysis and promotes oxidative phosphorylation. Furthermore, IL-10 suppresses mammalian target of rapamycin (mTOR) activity through the induction of an mTOR inhibitor, DDIT4. Consequently, IL-10 promotes mitophagy that eliminates dysfunctional mitochondria characterized by low membrane potential and a high level of reactive oxygen species. In the absence of IL-10 signaling, macrophages accumulate damaged mitochondria in a mouse model of colitis and inflammatory bowel disease patients, and this results in dysregulated activation of the NLRP3 inflammasome and production of IL-1ß.

The interferon gamma gene polymorphism +874 A/T is associated with severe acute respiratory syndrome.

BACKGROUND: Cytokines play important roles in antiviral action. We examined whether polymorphisms of IFN-gamma,TNF-alpha and IL-10 affect the susceptibility to and outcome of severe acute respiratory syndrome (SARS). METHODS: A case-control study was carried out in 476 Chinese SARS patients and 449 healthy controls. We tested the polymorphisms of IFN-gamma,TNF-alpha and IL-10 for their associations with SARS. RESULTS: IFN-gamma +874A allele was associated with susceptibility to SARS in a dose-dependent manner (P < 0.001). Individuals with IFN-gamma +874 AA and AT genotype had a 5.19-fold (95% Confidence Interval [CI], 2.78-9.68) and 2.57-fold (95% CI, 1.35-4.88) increased risk of developing SARS respectively. The polymorphisms of IL-10 and TNF-alpha were not associated with SARS susceptibility. CONCLUSION: IFN-gamma +874A allele was shown to be a risk factor in SARS susceptibility.

Macrophages monitor tissue osmolarity and induce inflammatory response through NLRP3 and NLRC4 inflammasome activation.

Interstitial osmolality is a key homeostatic variable that varies depending on the tissue microenvironment. Mammalian cells have effective mechanisms to cope with osmotic stress by engaging various adaptation responses. Hyperosmolality due to high dietary salt intake has been linked to pathological inflammatory conditions. Little is known about the mechanisms of sensing the hyperosmotic stress by the innate immune system. Here we report that caspase-1 is activated in macrophages under hypertonic conditions. Mice with high dietary salt intake display enhanced induction of Th17 response upon immunization, and this effect is abolished in caspase-1-deficient mice. Our findings identify an unknown function of the inflammasome as a sensor of hyperosmotic stress, which is crucial for the induction of inflammatory Th17 response.

Mannose-binding lectin enhances Toll-like receptors 2 and 6 signaling from the phagosome.

Innate immunity is the first-line defense against pathogens and relies on phagocytes, soluble components, and cell-surface and cytosolic pattern recognition receptors. Despite using hard-wired receptors and signaling pathways, the innate immune response demonstrates surprising specificity to different pathogens. We determined how combinatorial use of innate immune defense mechanisms defines the response. We describe a novel cooperation between a soluble component of the innate immune system, the mannose-binding lectin, and Toll-like receptor 2 that both specifies and amplifies the host response to Staphylococcus aureus. Furthermore, we demonstrate that this cooperation occurs within the phagosome, emphasizing the importance of engulfment in providing the appropriate cellular environment to facilitate the synergy between these defense pathways.

Mannose-binding lectin and susceptibility to infection in Chinese patients with systemic lupus erythematosus.

OBJECTIVE: To test the hypothesis that low serum mannose-binding lectin (MBL) levels, as a result of the single-nucleotide polymorphisms in the promoter region (-221 X/Y) and exon 1 (codon 54 A/B) of the MBL2 gene, predispose to infection in Chinese patients with systemic lupus erythematosus (SLE). METHODS: Two hundred forty-five patients with SLE were prospectively followed for the development of major infective episodes that required hospitalization and antibiotic treatment during 1992-2005. MBL genotypes were determined by polymerase chain reaction and serum MBL levels were measured by ELISA. RESULTS: In total, 254 major infections developed in 130 patients. Serum MBL levels were shown to correlate inversely with the number of bacterial infections (r = -0.13, p = 0.03). The distribution of MBL genotypes was similar in patients with and without major infection (p = 0.84). Patients with major infection also had more major lupus exacerbations that required daily prednisolone dose > or = 15 mg. Logistic regression showed that log MBL level (odds ratio 0.516, 95% confidence interval 0.305-0.873; p = 0.01) and major lupus exacerbation (OR 1.382, 95% CI 1.154-1.654; p < 0.001) were independent risk factors to major bacterial infection after adjustment for age and disease duration. Multiple regression analysis showed an increase in risk of bacterial infection by 34.2% for every decrease in serum MBL level by one log, and by 22.8% for each increase in number of major lupus exacerbations. CONCLUSION: Low serum MBL level predisposes Chinese patients with SLE to more major infections, in particular bacterial ones.

Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa.

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.

Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection.

Little is known about the innate immune response to severe acute respiratory syndrome (SARS) coronavirus (CoV) infection. Mannose-binding lectin (MBL), a key molecule in innate immunity, functions as an ante-antibody before the specific antibody response. Here, we describe a case-control study that included 569 patients with SARS and 1188 control subjects and used in vitro assays to investigate the role that MBL plays in SARS-CoV infection. The distribution of MBL gene polymorphisms was significantly different between patients with SARS and control subjects, with a higher frequency of haplotypes associated with low or deficient serum levels of MBL in patients with SARS than in control subjects. Serum levels of MBL were also significantly lower in patients with SARS than in control subjects. There was, however, no association between MBL genotypes, which are associated with low or deficient serum levels of MBL, and mortality related to SARS. MBL could bind SARS-CoV in a dose- and calcium-dependent and mannan-inhibitable fashion in vitro, suggesting that binding is through the carbohydrate recognition domains of MBL. Furthermore, deposition of complement C4 on SARS-CoV was enhanced by MBL. Inhibition of the infectivity of SARS-CoV by MBL in fetal rhesus kidney cells (FRhK-4) was also observed. These results suggest that MBL contributes to the first-line host defense against SARS-CoV and that MBL deficiency is a susceptibility factor for acquisition of SARS.