Prolonged immune alteration following resolution of acute inflammation in humans.

Acute inflammation is an immediate response to infection and injury characterised by the influx of granulocytes followed by phagocytosing mononuclear phagocytes. Provided the antigen is cleared and the immune system of the host is fully functional, the acute inflammatory response will resolve. Until now it is considered that resolution then leads back to homeostasis, the physiological state tissues experienced before inflammation occurred. Using a human model of acute inflammation driven by intradermal UV killed Escherichia coli, we found that bacteria and granulocyte clearance as well as pro-inflammatory cytokine catabolism occurred by 72h. However, following a lag phase of about 4 days there was an increase in numbers of memory T cells and CD163+ macrophage at the post-resolution site up to day 17 as well as increased biosynthesis of cyclooxygenase-derived prostanoids and DHA-derived D series resolvins. Inhibiting post-resolution prostanoids using naproxen showed that numbers of tissue memory CD4 cells were under the endogenous control of PGE2, which exerts its suppressive effects on T cell proliferation via the EP4 receptor. In addition, we re-challenged the post-resolution site with a second injection of E. coli, which when compared to saline controls resulted in primarily a macrophage-driven response with comparatively fewer PMNs; the macrophage-dominated response was reversed by cyclooxygenase inhibition. Re-challenge experiments were also carried out in mice where we obtained similar results as in humans. Therefore, we report that acute inflammatory responses in both humans and rodents do not revert back to homeostasis, but trigger a hitherto unappreciated sequence of immunological events that dictate subsequent immune response to infection.Wellcome Trust Senior Research Fellowship (Grant number: WT087520), Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant number: 107613/Z/15/Z) and the Barts Charity (Grant number: MGU0343)

A Comparison of Human Neutrophils Acquired from Four Experimental Models of Inflammation.

Defects in neutrophil function have been implicated in a wide spectrum of clinical conditions. Several models are employed to study activated human neutrophils akin to those found at a site of inflammation. These include whole blood (WB) ex vivo stimulation with lipopolysaccharide (LPS) and in vivo techniques: cantharidin blister, skin windows and intra-dermal injection of UV-killed E.coli (UVKEc). Neutrophils obtained from these have never been compared. We compared the activation status of neutrophils from each technique in order to inform the optimal model for use in human studies. Healthy male volunteers were randomised to undergo one of the four techniques (n = 5/group). LPS: WB stimulated with 1ng/ml of LPS for 4 hours. Cantharidin: 12.5μl of 0.1% cantharidin elicited a single blister, aspirated at 24 hours. Skin windows: four 6mm mechanical-suction blisters created, de-roofed and an exudate-collection chamber placed over the windows for 4 hours before aspiration. UVKEc: 1.5 x 107 UVKEc injected intra-dermally. A single 10mm mechanical-suction blister formed and aspirated at 4 hours. Unstimulated WB used as the control. Flow cytometry was used to determine activation status using CD16, CD11b, CD54, CD62L and CD88. Functional status was assessed with a phagocytosis assay. The pattern of neutrophil activation was similar in all models. Neutrophil CD11b was elevated in all models, most markedly in UVKEc (p<0.0001), and CD54 was also elevated but only significant in the LPS model (p = 0.001). CD62L was significantly reduced in all 4 models (p<0.0001) and CD88 was also suppressed in all. There were no changes in CD16 in any model, neither was there any significant difference in the phagocytic capacity of the neutrophils. In summary, there are no significant differences in activation marker expression or phagocytic capacity in the neutrophils obtained from each technique. Therefore we believe whole blood stimulation is the best model in experimentally challenging inpatient populations

Acute inflammatory response to intradermal UVkEc injection.

<p>Acute inflammation was triggered in the ventral aspect of forearm of healthy volunteers by the intradermal injection of 1.5 x 10⁷ UV-killed <i>E</i>. <i>coli</i> (UVKEc) suspended in 100 μl of sterile saline. Vascular hyperaemia at the site was assessed by laser Doppler imager and the representative flux images after a specified time point are shown here <b>(A)</b>. A 3 mm skin punch biopsy was taken from the inflamed site under local anaesthesia at the specified interval and formalin fixed paraffin embedded (FFPE) skin sections were probed by immunohistochemistry for <i>E</i>. <i>coli</i> LPS. The representative sections are shown here <b>(B)</b>. A suction blister was raised over the inflamed site at the specified interval to collect the inflammatory exudate. Exudate was centrifuged to separate cells from the supernatant. The immune cell subsets were identified by polychromatic flow cytometry. The total count/ml of neutrophils in the inflammatory exudate at specified interval is shown here <b>(C)</b>. IL-6, TNFα and IL1β in the cell free exudate were measured using multiplex ELISA and their concentrations in the exudate at the specified intervals are shown here <b>(D-F, respectively)</b>. n = 3 for each time point. Data presented as mean ± SEM.</p

Post-resolution tissue accumulation of macrophages.

<p>Acute inflammation was triggered in the ventral aspect of forearm of healthy volunteers by the intradermal injection of 1.5 x 10⁷ UV-killed <i>E</i>. <i>coli</i> (UVKEc) suspended in 100 μl of sterile saline. A suction blister was raised over the inflamed site at the specified interval to collect the inflammatory exudate. Exudate was centrifuged to separate cells from the supernatant. The immune cell subsets were identified by polychromatic flow cytometry. The numbers of CD14⁺ mononuclear cells at the specified interval are shown here <b>(A)</b>. A 3mm skin punch biopsy was taken from the inflamed site under local anaesthesia at the specified interval and the formalin fixed paraffin embedded (FFPE) skin sections were probed by immunohistochemistry for CD163. The representative sections are shown here <b>(B).</b> MCP-1, IP-10, MDC and MCP-4 <b>(panels C-F)</b> in the cell free exudate were measured using multiplex ELISA and their concentrations in the exudate at the specified intervals are shown here <b>(C)</b>. n = 3 for each time point. Data presented as mean ± SEM.</p

Increased lipid mediator biosynthesis during post-resolution biology.

<p>Acute inflammation was triggered in the ventral aspect of forearm of healthy volunteers by the intradermal injection of 1.5 x 10⁷ UV-killed <i>E</i>. <i>coli</i> (UVKEc) suspended in 100 μl of sterile saline. A suction blister was raised over the inflamed site at the specified interval to collect the inflammatory exudate. Lipid mediators in the cell free exudate were analysed by liquid chromatography tandem mass spectrophotometry (LC MS/MS). The levels of PGE₂ <b>(A)</b>, TXB₂ <b>(B),</b> PGD₂ <b>(C),</b> PGF_2α <b>(D),</b> LXB₄ <b>(E)</b>, 5,15-diHETE <b>(F)</b>, RvD5 <b>(G)</b>, 5,15 diHETE (G) and RvE3 <b>(H)</b> at specified interval are shown here. n = 3 for each time point. Data presented as mean ± SEM.</p

Post-resolution tissue accumulation of memory T cells and lymphocyte mitogens.

<p>Acute inflammation was triggered in the ventral aspect of forearm of healthy volunteers by the intradermal injection of 1.5 x 10⁷ UV-killed <i>E</i>. <i>coli</i> (UVKEc) suspended in 100 μl of sterile saline. A suction blister was raised over the inflamed site at the specified interval to collect the inflammatory exudate. Exudate was centrifuged to separate cells from the supernatant. The immune cell subsets were identified by polychromatic flow cytometry. The numbers of CD4⁺/CD45RO⁺/CCR7^- <b>(A),</b> CD8⁺/CD45RO⁺/CCR7^- memory T cells <b>(B)</b> and CD56⁺ NK cells <b>(C)</b> at the specified interval are shown here. IL-15 <b>(D)</b> and IL-7 <b>(E)</b> in the cell free exudate were measured using multiplex ELISA and their concentrations in the exudate at the specified intervals are shown here. n = 3 for each time point. Data presented as mean ± SEM.</p

Inhibition of post-resolution prostanoids increases numbers of local memory CD4⁺ T cells.

<p>Acute inflammation was triggered in the ventral aspect of forearm of healthy volunteers by the intradermal injection of 1.5 x 10⁷ UV-killed <i>E</i>. <i>coli</i> (UVKEc) suspended in 100 μl of sterile saline. Inflammation was allowed to progress for 9 days after which volunteers were administered naproxen (500 mg, BD) orally for six days to block the rise in post-resolution prostanoids. On day 14 suction blister were elicited to enumerate cell profiles focusing on CD4⁺/CD45RO⁺/CCR7^- <b>(A)</b> and CD8⁺/CD45RO⁺/CCR7^- memory T cells <b>(B).</b> To determine what effect prostanoids have on memory T cell function, peripheral blood CD3⁺ cells were isolated from chicken pox-exposed individuals and incubated with varicella zoster antigen with/without PGE₂ at concentrations found in post-resolution tissue <b>(C)</b> with/without EP 2/4 antagonists <b>(D)</b> while CD3⁺ cells from chicken pox naïve individuals were used as negative controls <b>(E)</b>. n = 3 for each time point. Data presented as mean ± se. *<i>p</i>≤0.05, **<i>p</i>≤0.01; as determined by ANOVA followed by Bonferroni t test or by two-tailed Student's t test.</p

Inflammatory Resolution Triggers a Prolonged Phase of Immune Suppression through COX-1/mPGES-1-Derived Prostaglandin E₂

Acute inflammation is characterized by granulocyte infiltration followed by efferocytosing mononuclear phagocytes, which pave the way for inflammatory resolution. Until now, it was believed that resolution then leads back to homeostasis, the physiological state tissues experience before inflammation occurred. However, we discovered that resolution triggered a prolonged phase of immune suppression mediated by prostanoids. Specifically, once inflammation was switched off, natural killer cells, secreting interferon γ (IFNγ), infiltrated the post-inflamed site. IFNγ upregulated microsomal prostaglandin E synthase-1 (mPGES-1) alongside cyclo-oxygenase (COX-1) within macrophage populations, resulting in sustained prostaglandin (PG)E2 biosynthesis. Whereas PGE2 suppressed local innate immunity to bacterial infection, it also inhibited lymphocyte function and generated myeloid-derived suppressor cells, the net effect of which was impaired uptake/presentation of exogenous antigens. Therefore, we have defined a sequence of post-resolution events that dampens the propensity to develop autoimmune responses to endogenous antigens at the cost of local tissue infection

Characterisation of Leukocytes in a Human Skin Blister Model of Acute Inflammation and Resolution

There is an increasing need to understand the leukocytes and soluble mediators that drive acute inflammation and bring about its resolution in humans. We therefore carried out an extensive characterisation of the cantharidin skin blister model in healthy male volunteers. A novel fluorescence staining protocol was designed and implemented, which facilitated the identification of cell populations by flow cytometry. We observed that at the onset phase, 24 h after blister formation, the predominant cells were CD16hi/CD66b+ PMNs followed by HLA-DR+/CD14+ monocytes/macrophages, CD11c+ and CD141+ dendritic cells as well as Siglec-8+ eosinophils. CD3+ T cells, CD19+ B cells and CD56+ NK cells were also present, but in comparatively fewer numbers. During resolution, 72 h following blister induction, numbers of PMNs declined whilst the numbers of monocyte/macrophages remain unchanged, though they upregulated expression of CD16 and CD163. In contrast, the overall numbers of dendritic cells and Siglec-8+ eosinophils increased. Post hoc analysis of these data revealed that of the inflammatory cytokines measured, TNF-α but not IL-1β or IL-8 correlated with increased PMN numbers at the onset. Volunteers with the greatest PMN infiltration at onset displayed the fastest clearance rates for these cells at resolution. Collectively, these data provide insight into the cells that occupy acute resolving blister in humans, the soluble mediators that may control their influx as well as the phenotype of mononuclear phagocytes that predominate the resolution phase. Further use of this model will improve our understanding of the evolution and resolution of inflammation in humans, how defects in these over-lapping pathways may contribute to the variability in disease longevity/chronicity, and lends itself to the screen of putative anti-inflammatory or pro-resolution therapies