Exposure to normobaric hypoxia shapes the acute inflammatory response in human whole blood cells in vivo.

Cells of the immune defence, especially leukocytes, often have to perform their function in tissue areas that are characterized by oxygen deficiency, so-called hypoxia. Physiological hypoxia significantly affects leukocyte function and controls the innate and adaptive immune response mainly through transcriptional gene regulation via the hypoxia-inducible factors (HIFs). Multiple pathogens including components of bacteria, such as lipopolysaccharides (LPS) trigger the activation of leukocytes. HIF pathway activation enables immune cells to adapt to both hypoxic environments in physiological and inflammatory settings and modulates immune cell responses through metabolism changes and crosstalk with other immune-relevant signalling pathways. To study the mutual influence of both processes in vivo, we used a human endotoxemia model, challenging participants with an intravenous LPS injection post or prior to a 4-h stay in a hypoxic chamber with normobaric hypoxia of 10.5% oxygen. We analysed changes in gene expression in whole blood cells and determined inflammatory markers to unveil the crosstalk between both processes. Our investigations showed differentially altered gene expression patterns of HIF and target genes upon in vivo treatment with LPS and hypoxia. Further, we found evidence for effects of hypoxic priming upon inflammation in combination with immunomodulatory effects in whole blood cells in vivo. Our work elucidates the complex interplay of hypoxic and inflammatory HIF regulation in human immune cells and offers new perspectives for further clinical research.

Acute hypoxic conditions preceding endotoxin administration result in an increased proinflammatory cytokine response in healthy men.

Tissues often experience hypoxia at sites of inflammation due to malperfusion, massive immune cell recruitment, and increased oxygen consumption. Organisms adapt to these hypoxic conditions through the transcriptional activation of various genes. In fact, there is significant crosstalk between the transcriptional responses to hypoxia and inflammatory processes. This interaction, named inflammatory hypoxia, plays a crucial role in various diseases including malignancies, chronic inflammatory lung diseases, and sepsis. To further elucidate the crosstalk between hypoxia and inflammation in vivo and assess its potential for innovative therapies, our study aimed at investigating the impact of acute hypoxic conditions on inflammation-induced immune responses. To this end, we exposed healthy human subjects to hypoxia either before (hypoxia priming) or after a single intravenous (i.v.) injection of 0.4 ng/kg LPS. Our data show that hypoxia exposure prior to LPS injection (hypoxia priming) amplified the proinflammatory response. This was reflected by an increase in body temperature, plasma noradrenaline levels, and the production of proinflammatory cytokines (i.e., IL-6 and TNF-α), compared with LPS control conditions. These effects were not observed when participants were exposed to hypoxia after LPS administration, demonstrating that the interaction between hypoxia and inflammation highly depends on the timing of both stimuli. Our findings suggest that acute hypoxia (i.e., hypoxia priming) modulates transient inflammation, leading to an enhanced proinflammatory response in healthy human subjects. This highlights the need for further investigations to understand the pathology of various hypoxia-inducible factor (HIF)-associated inflammatory diseases and to develop suitable, innovative therapies.NEW & NOTEWORTHY To our knowledge, this is the first in vivo study investigating the effects of hypoxia preceding (hypoxia priming) or following LPS administration on the endotoxin-induced inflammatory response in healthy human subjects. The data show that hypoxia priming amplified the proinflammatory response, reflected by an increased body temperature, increased plasma noradrenaline levels, and higher production of proinflammatory cytokines (i.e., IL-6 and TNF-α) compared with LPS control conditions.

Amplified gut feelings under inflammation and depressed mood: A randomized fMRI trial on interoceptive pain in healthy volunteers.

BACKGROUND: Inflammation and depressed mood constitute clinically relevant vulnerability factors for enhanced interoceptive sensitivity and chronic visceral pain, but their putative interaction remains untested in human mechanistic studies. We tested interaction effects of acute systemic inflammation and sad mood on the expectation and experience of visceral pain by combining experimental endotoxemia with a mood induction paradigm. METHODS: The double-blind, placebo-controlled, balanced crossover fMRI-trial in N = 39 healthy male and female volunteers involved 2 study days with either intravenous administration of low-dose lipopolysaccharide (LPS, 0.4 ng/kg body weight; inflammation condition) or saline (placebo condition). On each study, day two scanning sessions were conducted in an experimentally induced negative (i.e., sad) and in a neutral mood state, accomplished in balanced order. As a model of visceral pain, rectal distensions were implemented, which were initially calibrated to be moderately painful. In all sessions, an identical series of visceral pain stimuli was accomplished, signaled by predictive visual conditioning cues to assess pain anticipation. We assessed neural activation during the expectation and experience of visceral pain, along with unpleasantness ratings in a condition combining an inflammatory state with sad mood and in control conditions. All statistical analyses were accomplished using sex as covariate. RESULTS: LPS administration led to an acute systemic inflammatory response (inflammation X time interaction effects for TNF-α, IL-6, and sickness symptoms, all p <.001). The mood paradigm effectively induced distinct mood states (mood X time interaction, p <.001), with greater sadness in the negative mood conditions (both p <.001) but no difference between LPS and saline conditions. Significant main and interaction effects of inflammation and negative mood were observed for pain unpleasantness (all p <.05). During cued pain anticipation, a significant inflammation X mood interaction emerged for activation of the bilateral caudate nucleus and right hippocampus (all pFWE < 0.05). Main effects of both inflammation and mood were observed in multiple regions, including insula, midcingulate cortex, prefrontal gyri, and hippocampus for inflammation, and midcingulate, caudate, and thalamus for mood (all pFWE < 0.05). CONCLUSIONS: Results support an interplay of inflammation and sad mood on striatal and hippocampal circuitry engaged during visceral pain anticipation as well as on pain experience. This may reflect a nocebo mechanism, which may contribute to altered perception and interpretation of bodily signals. At the interface of affective neuroscience and the gut-brain axis, concurrent inflammation and negative mood may be vulnerability factors for chronic visceral pain.

Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes.

BACKGROUND: CD19+CD24hiCD38hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus. METHODS: PBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA. RESULTS: The amount of CD19+ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%. CONCLUSION: Calcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.

Pharmacodynamic monitoring of mammalian target of rapamycin inhibition by phosphoflow cytometric determination of p70S6 kinase activity.

BACKGROUND: Immunosuppressive therapy with mammalian target of rapamycin inhibitors (mTORi) requires maintenance of an effective inhibition of the alloimmune response, whereas reducing drug-related nephrotoxicity. Therapeutic monitoring is based on mTORi trough levels, which do not necessarily reflect biologic effects on the PI3K-Akt-mTOR pathway and hence may often result in under-immunosuppression or over-immunosuppression. METHODS: Phosphorylation of p70S6 kinase was studied by phosphoflow cytometry and by Western blot in both peripheral blood monocyte cells and CD3+ T cells of renal transplant recipients (RTX) receiving tacrolimus (n=34) or cyclosporine A (CsA) (n=24) or an mTORi (n=26). 16 healthy age-matched volunteers served as a control group. To clarify whether p70S6K activity is varying among CD4(+) T-cell subsets, cell sorted CD4(+)CD25(hi) regulatory T cells (Tregs) and CD4(+)CD25- T cells were analyzed for p70S6K phosphorylation. RESULTS: Simultaneous analysis of p70S6K phosphorylation by phosphoflow cytometry and Western blot showed high correlation in peripheral blood mononuclear cells of renal transplant patients (r=0.91, P<0.001). Mammalian target of rapamycin inhibition was associated with marked reduction of p70S6K phosphorylation compared to healthy volunteers or RTX patients receiving calcineurin inhibitors (all P<0.001) but did not correlate with mTORi trough levels. Interleukin-2 production in mitogen-stimulated CD3(+) T cells correlated with the degree of p70S6K phosphorylation in everolimus-treated patients. p70S6K phosphorylation in CD4+CD25(hi) Tregs was significantly lower compared to CD4(+)CD25- T cells (n=3). In mTORi treated RTX recipients, p70S6K phosphorylation was selectively reduced in CD4(+)CD25- T cells leaving CD4(+)CD25(hi) Tregs unimpaired. CONCLUSION: Phosphoflow cytometric quantification of p70S6K phosphorylation may play an adjunct role to pharmacodynamically guide an individualized mTORi therapy. It may have potential to be used in purity testing of Treg suspensions generated for adoptive tolerogenic therapies.