Madhur P. Motwani

Resolution of acute inflammation bridges the gap between innate and adaptive immunity.

Acute inflammation is traditionally characterized by polymorphonuclear leukocytes (PMN) influx followed by phagocytosing macrophage (Mφs) that clear injurious stimuli leading to resolution and tissue homeostasis. However, using the peritoneal cavity, we found that although innate immune-mediated responses to low-dose zymosan or bacteria resolve within days, these stimuli, but not hyperinflammatory stimuli, trigger a previously overlooked second wave of leukocyte influx into tissues that persists for weeks. These cells comprise distinct populations of tissue-resident Mφs (resMφs), Ly6c(hi) monocyte-derived Mφs (moMφs), monocyte-derived dendritic cells (moDCs), and myeloid-derived suppressor cells (MDSCs). Postresolution mononuclear phagocytes were observed alongside lymph node expansion and increased numbers of blood and peritoneal memory T and B lymphocytes. The resMφs and moMφs triggered FoxP3 expression within CD4 cells, whereas moDCs drive T-cell proliferation. The resMφs preferentially clear apoptotic PMNs and migrate to lymph nodes to bring about their contraction in an inducible nitric oxide synthase-dependent manner. Finally, moMφs remain in tissues for months postresolution, alongside altered numbers of T cells collectively dictating the magnitude of subsequent acute inflammatory reactions. These data challenge the prevailing idea that resolution leads back to homeostasis and asserts that resolution acts as a bridge between innate and adaptive immunity, as well as tissue reprogramming.

Macrophage development and polarization in chronic inflammation

The mononuclear phagocyte system consists of monocytes, acrophages and dendritic cells. The main functions of monocyte nd macrophages include chemotaxis, phagocytosis, endocytois, secretion of factors that modulate inflammatory responses nd microbial killing; all of which are integral to homeostasis, mmune defence and tissue repair [1]. In humans, monocytes were nitially defined on the basis of morphology (size and density) s well as intracellular monocyte-specific esterase, for instance. xpression level of CD64 (Fc RI) was used for some time where D64+ cells were larger and more phagocytic than smaller CD64− ells, which preferentially expressed MHC-I/II and secreted type 1 nterferons. Nowadays, flow cytometry using anti-CD14 (directed o the LPS-binding domain) and CD16 (Fc RIII-binding domain) ntibodies has given three classifications namely, CD14++/CD16− classical), CD14++/CD16+ (intermediate) and CD14low/CD16++ non-classical) [2,3]. Typically, CD14++ monocytes (classical and ntermediate) represent 90% of blood monocytes and are responible for phagocytosis, reactive oxygen species production as well s pro-inflammatory cytokine production in response to LPS via classical MyD88-, p38-, and NFB dependent pathway. Within his subset, CD14++/CD16− cells are responsible for the bulk of OS, IL-8, and IL-6 production whilst the CD14++/CD16+ subet produce TNF and IL-1, in response to LPS [4]. In contrast, on-classical monocytes, which constitute the remaining 5-10% onocytes, are less phagocytic compared to CD14++/CD16− cells nd do not produce appreciable levels of reactive oxygen species, ysozyme or cytokines in response to cell-surface Toll-like receptor TLR1, 2 and 4) agonists. Instead, these CD14low/CD16++ nonlassical monocytes selectively secrete TNF , IL-1 and CCL3 in

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.

Novel translational model of resolving inflammation triggered by UV-killed E. coli.

Whilst numerous studies investigating the aetiology of inflammatory diseases have been performed in rodents, the applicability of these data to human pathophysiology is frequently debated. Regardless of the strengths and weaknesses of rodent models in biomedical research, there is a need to develop models of experimental inflammation in humans. Here, we describe a self‐resolving acute inflammatory response triggered by the intradermal injection of UV‐killed Escherichia coli into the forearm of healthy volunteers. Cells and exudates were harvested from onset to resolution by applying negative pressure over the inflamed site. Onset was characterized by high blood flow, neutrophilia and peak levels of pro‐inflammatory cytokines, whilst resolution showed a decline in blood blow, reduction in neutrophils, increase in monocytes/macrophages and waning of classic pro‐inflammatory cytokine levels. An anti‐inflammatory effect, defined as suppression of onset phase events, was demonstrated by administering naproxen, a conventional non‐steroidal anti‐inflammatory drug. In summary, this model of resolving acute inflammation is minimally invasive, highly tractable and allows simultaneous investigation of the vascular response, cellular trafficking and chemical mediator profile of onset and resolution phases of acute inflammation in humans. It can serve as a translational platform to provide mechanistic insights and to test the clinical efficacy of novel anti‐inflammatory and pro‐resolving drugs, and also as a tool in patients to explore inherent defects in resolution pathways.

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

Summary 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.

Potent Anti‐Inflammatory and Pro‐Resolving Effects of Anabasum in a Human Model of Self‐Resolving Acute Inflammation

Anabasum is a synthetic analog of Δ8‐tetrahydrocannabinol (THC)‐11‐oic acid that in preclinical models of experimental inflammation exerts potent anti‐inflammatory actions with minimal central nervous system (CNS) cannabimimetic activity. Here we used a novel model of acute inflammation driven by i.d. UV‐killed E. coli in healthy humans and found that anabasum (5 mg) exerted a potent anti‐inflammatory effect equivalent to that of prednisolone in terms of inhibiting neutrophil infiltration, the hallmark of acute inflammation. These effects arose from the inhibition of the neutrophil chemoattractant LTB4, while the inhibition of antiphagocytic prostanoids (PGE2, TxB2, and PGF2α) resulted in enhanced clearance of inflammatory stimulus from the injected site. Anabasum at the higher dose of 20 mg possessed the additional properties of triggering the biosynthesis of specialized pro‐resolving lipid mediators including LXA4, LXB4, RvD1, and RvD3. Collectively, we demonstrate for the first time a striking anti‐inflammatory and pro‐resolution effects of a synthetic analog of THC in healthy humans.

Pro-resolving mediators promote resolution in a human skin model of UV-killed Escherichia coli-driven acute inflammation.

While the treatment of inflammatory disorders is generally based on inhibiting factors that drive onset of inflammation, these therapies can compromise healing (NSAIDs) or dampen immunity against infections (biologics). In search of new antiinflammatories, efforts have focused on harnessing endogenous pathways that drive resolution of inflammation for therapeutic gain. Identification of specialized pro-resolving mediators (SPMs) (lipoxins, resolvins, protectins, maresins) as effector molecules of resolution has shown promise in this regard. However, their action on inflammatory resolution in humans is unknown. Here, we demonstrate using a model of UV-killed Escherichia coli–triggered skin inflammation that SPMs are biosynthesized at the local site at the start of resolution, coinciding with the expression of receptors that transduce their actions. These include receptors for lipoxin A4 (ALX/FPR2), resolvin E1 (ChemR23), resolvin D2 (GPR18), and resolvin D1 (GPR32) that were differentially expressed on the endothelium and infiltrating leukocytes. Administering SPMs into the inflamed site 4 hours after bacterial injection caused a reduction in PMN numbers over the ensuing 6 hours, the phase of active resolution in this model. These results indicate that in humans, the appearance of SPMs and their receptors is associated with the beginning of inflammatory resolution and that their therapeutic supplementation enhanced the resolution response.

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.

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.