In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

/pdf/the-fate-and-lifespan-of-human-monocyte-subsets-in-steady-24apxmwqad.pdf

The fate and lifespan of human monocyte subsets in steady state and systemic inflammation.

Background 
Sepsis is defined as a dysregulated immune response to infection. Impaired immune response in sepsis, often described as endotoxin tolerance, is characterized by unresponsiveness of monocytes on lipopolysaccharide (LPS) stimulation to release tumor necrosis factor α (TNFα). Furthermore, decreased monocyte surface protein expression of human leucocyte antigen DR (HLA-DR) is a marker for changes of the innate immune response during sepsis. Quantitative polymerase chain reaction (qPCR) and flow-cytometry (FACS) have been used to measure protein or gene expression of HLA-DR. We aimed to determine whether changes in mRNA expression of HLA-DR are associated with impaired TNFα response in human sepsis.


Methods 
Surface protein together with mRNA expression of HLA-DR were measured by FACS and qPCR in a cohort of 9 sepsis patients and compared to 10 pre-operative control patients in a prospective study. In addition, 20 patients with post-surgical inflammation, 20 patients with sepsis or septic shock were included and TNFα was determined following ex vivo stimulation of whole blood with 500 pg/mL LPS. Total RNA was prepared from whole blood and subjected to qPCR analysis for expression analysis of HLA-DR alpha (HLA-DRA) to correlate TNFα response with HLA-DRA expression.


Results 
Patients with sepsis presented higher numbers of monocytes in peripheral blood (P<0.001) but decreased surface protein and mRNA HLA-DR levels when compared to controls. In all patients mRNA expression of HLA-DRA was decreased by approximately 70% compared to controls (P<0.01) and was lowest in patients with sepsis or septic shock (P<0.01). TNFα response to LPS was decreased in all patients (median 319 pg/mL versus controls 1256 pg/mL; P<0.01) and lowest in patients with sepsis or septic shock (median 128 pg/mL; P<0.01). HLA-DRA correlated positively with TNFα response in all study participants (r +0.60, P<0.001) and within patients (r +0.67, P<0.001). The TNFα:HLA-DRA ratio correlated negatively with severity and the Sequential Organ Failure Assessment (SOFA) score (Spearman’s rho -0.59, P<0.001)


Conclusion 
In this study, HLA-DRA expression was associated with a functional assay of the innate immune response. Future interventional studies aimed at the immune response during sepsis could make use of these methods for optimizing target groups based on biological plausibility and intervention effectiveness.

/pdf/human-leucocyte-antigen-hla-dr-gene-expression-is-reduced-in-3mtc38tufm.pdf

Human leucocyte antigen (HLA-DR) gene expression is reduced in sepsis and correlates with impaired TNFα response: A diagnostic tool for immunosuppression?

Identifying patients in the immunosuppressive phase of sepsis is essential for development of immunomodulatory therapies. Little data exists comparing the ability of the two most well-studied markers of sepsis-induced immunosuppression, human leukocyte antigen (HLA)-DR expression and lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-ɑ) production, to predict mortality and morbidity. The purpose of this study was to compare HLA-DR expression and LPS-induced TNF-ɑ production as predictors of 28-day mortality and acquisition of secondary infections in adult septic patients. A single-center, prospective observational study of 83 adult septic patients admitted to a medical or surgical intensive care unit. Blood samples were collected at three time points during the septic course (days 1–2, days 3–4, and days 6–8 after sepsis diagnosis) and assayed for HLA-DR expression and LPS-induced TNF-ɑ production. A repeated measures mixed model analysis was used to compare values of these immunological markers among survivors and non-survivors and among those who did and did not develop a secondary infection. Twenty-five patients (30.1 %) died within 28 days of sepsis diagnosis. HLA-DR expression was significantly lower in non-survivors as compared to survivors on days 3–4 (p = 0.04) and days 6–8 (p = 0.002). The change in HLA-DR from days 1–2 to days 6–8 was also lower in non-survivors (p = 0.04). Median HLA-DR expression decreased from days 1–2 to days 3–4 in patients who developed secondary infections while it increased in those without secondary infections (p = 0.054). TNF-ɑ production did not differ between survivors and non-survivors or between patients who did and did not develop a secondary infection. Monocyte HLA-DR expression may be a more accurate predictor of mortality and acquisition of secondary infections than LPS-stimulated TNF-ɑ production in adult medical and surgical critically ill patients.

/pdf/comparison-of-monocyte-human-leukocyte-antigen-dr-expression-2r63mtjycf.pdf

Comparison of Monocyte Human Leukocyte antigen-DR Expression and Stimulated Tumor Necrosis Factor Alpha Production as Outcome Predictors in Severe Sepsis: A Prospective Observational Study

The concept of the “leaky gut” has received increasing attention in the lay press and also in the scientific literature of late due to its associations with numerous gastrointestinal (GI) and non-GI diseases such as irritable bowel syndrome, Alzheimer’s disease, asthma, type 2 diabetes, hepatic steatosis, and many others [1] to the point that dietary modifications, probiotics, and other interventions intended to increase “gut integrity” are recommended as treatments for a host of diseases [2, 3]. The premise underlying the “leaky gut” hypothesis is that physiologic stressors such as anxiety, intensive exercise, or dietary components such as emulsifiers increase intestinal mucosal paracellular permeability, enhancing entry of pathogenic bacteria and bacterial toxins into the systemic circulation, provoking systemic inflammation and triggering numerous diseases. Although data strongly support the concept that bacterial endotoxin (lipopolysaccharide; LPS) is pro-inflammatory and that inflammation can increase intestinal paracellular permeability, few convincing data obtained form the study of intact intestinal tissue support paracellular transport of bacteria and bacterial toxins from lumen to submucosa. In this perspective, we will review the data regarding intestinal paracellular transport, providing views based on the preponderance of the available data regarding the mechanisms of intestinal transport of solutes, bacteria, and bacterial toxins in relation to paracellular permeability with the primary objective of contrasting the intestinal transport pathways for intact bacteria and bacterial toxins such as LPS with the paracellular pathways by which ions and small organic molecules are transported across the mucosa. In this fashion, we hope to provide data that will support our conclusion that the intestinal paracellular space is a major route of transport of water and small solutes such as ions and small soluble organic molecules between the lumen and submucosal space, and not a means by which large molecules, lipophilic substances, or macromolecular structures such as proteins, particulate matter, or intact bacteria are absorbed. We hope also to further the understanding of the intestinal transport of bacteria and bacterial toxins, in particular since such translocation underlies many important and highly morbid diseases such as sepsis, the systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) [4, 5].

/pdf/the-leaky-gut-tight-junctions-but-loose-associations-2l4iqd9eqh.pdf

The “Leaky Gut”: Tight Junctions but Loose Associations?

In critically ill patients, organ dysfunctions are routinely assessed, monitored, and treated. Mounting data show that substantial critical illness-induced changes in the immune system can be observed in most ICU patients and that not only "hyper-inflammation" but also persistence of an anti-inflammatory phenotype (as in sepsis-associated immunosuppression) is associated with increased morbidity and mortality. Despite common perception, changes in functional immunity cannot be adequately assessed by routine inflammatory biomarkers such as C-reactive protein, procalcitonin, or numerical analysis of leukocyte (sub)-counts. Cytokines appear also not suited due to their short half-life and pleiotropy, their unexclusive origin from immune cells, and their potential to undergo antagonization by circulating inactivating molecules. Thus, beyond leukocyte quantification and use of routine biomarkers, direct assessment of immune cell function seems required to characterize the immune systems' status. This may include determination of, e.g., ex vivo cellular cytokine release, phagocytosis activity, and/or antigen-presenting capacity. In this regard, standardized flow-cytometric assessment of the major histocompatibility-II complex human leukocyte antigen (-D related) (HLA-DR) has gained particular interest. Monocytic HLA-DR (mHLA-DR) controls the interplay between innate and adaptive immunity and may serve as a "global" biomarker of injury-associated immunosuppression, and its decreased expression is associated with adverse clinical outcomes (e.g., secondary infection risk, mortality). Importantly, recent data demonstrate that injury-associated immunosuppression can be reversed-opening up new therapeutic avenues in affected patients. Here we discuss the potential scientific and clinical value of assessment of functional immunity with a focus on monocytes/macrophages and review the current state of knowledge and potential perspectives for affected critically ill patients.

/pdf/assessment-of-immune-organ-dysfunction-in-critical-illness-3d5ta9ce7g.pdf

Assessment of immune organ dysfunction in critical illness: utility of innate immune response markers

Objective:
Reduced ex vivo lipopolysaccharide (LPS) stimulated whole blood pro-inflammatory cytokine release is a hallmark of immunosuppression in the critically ill and predicts adverse clinical outcomes. No standard technique for performing the assay currently exists. The impact of methodological heterogeneity was determined.

/pdf/stimulated-whole-blood-cytokine-release-as-a-biomarker-of-32wisye25z.pdf

Stimulated Whole Blood Cytokine Release as a Biomarker of Immunosuppression in the Critically Ill: The Need for a Standardized Methodology

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances. Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a 'sepsis-like' state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

https://www.jove.com/pdf/53913/jove-protocol-53913-intravenous-endotoxin-challenge-healthy-humans-an-experimental

Elisabetta Segre

Papers

Stimulated Whole Blood Cytokine Release as a Biomarker of Immunosuppression in the Critically Ill: The Need for a Standardized Methodology

Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation.

Crack-use-associated pneumonia: a case report