Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Specific macrophage subsets accumulate in human subcutaneous and omental fat depots during obesity.

Obesity is a chronic inflammatory disease associated with adipose tissue macrophage (ATM) activation. ATMs from lean mice contribute to tissue homeostasis by their M2-oriented polarization, whereas obesity leads to an increase of M1 inflammatory ATMs that underlies obesity-related metabolic disorders. In humans, studies characterizing ATMs and their functional status are limited. Here we investigated ATM phenotype in visceral (VAT) and subcutaneous (SAT) adipose tissue from healthy lean and obese individuals using two molecules previously identified as markers of M1-like and M2-like/tissue-resident macrophages, the C-type lectin CLEC5A and the scavenger receptor CD163L1, respectively. CD163L1 was expressed by the majority of ATMs, and CD163L1+ ATM density was greater with respect to cells expressing the pan-macrophage markers CD68 or CD11b. ATM counts in SAT, but not in VAT, increased in obese compared to lean individuals, measured with the three markers. Accordingly, CD163L1, CD68 and ITGAM gene expression was significantly enhanced in obese with respect to control individuals only in SAT. CLEC5A+ ATMs had a proinflammatory profile and were abundant in the lean VAT, but their density diminished in obesity. The only ATM subset that increased its counts in the obese VAT had a mixed M1-like (CD11c+ CD163- CD209- ) and M2-like (CLEC5A- CD206+ ) phenotype. ATM expansion was dominated by a subset of M2-like macrophages (CD11c- CLEC5A- CD163+ CD206+ CD209+ ) in the obese SAT, with a minor contribution of a CD11c+ CLEC5A- ATM subpopulation. Thus, both SAT and VAT seems to limit inflammation during obesity by differentially altering their ATM subset composition.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons.

OBJECTIVES: Vagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human. DESIGN: Using Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1-5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI. RESULTS: EFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery. CONCLUSION: Enteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI. TRIAL REGISTRATION NUMBER: NCT02425774.

Atypical Activin A and IL-10 Production Impairs Human CD16+ Monocyte Differentiation into Anti-Inflammatory Macrophages.

Human CD14(++)CD16(-) and CD14(+/lo)CD16(+) monocyte subsets comprise 85 and 15% of blood monocytes, respectively, and are thought to represent distinct stages in the monocyte differentiation pathway. However, the differentiation fates of both monocyte subsets along the macrophage (Mϕ) lineage have not yet been elucidated. We have now evaluated the potential of CD14(++) CD16(-) and CD16(+) monocytes to differentiate and to be primed toward pro- or anti-inflammatory Mϕs upon culture with GM-CSF or M-CSF, respectively (subsequently referred to as GM14, M14, GM16, or M16). Whereas GM16 and GM14 were phenotypic and functionally analogous, M16 displayed a more proinflammatory profile than did M14. Transcriptomic analyses evidenced that genes associated with M-CSF-driven Mϕ differentiation (including FOLR2, IL10, IGF1, and SERPINB2) are underrepresented in M16 with respect to M14. The preferential proinflammatory skewing of M16 relative to M14 was found to be mediated by the secretion of activin A and the low levels of IL-10 produced by M16. In fact, activin A receptor blockade during the M-CSF-driven differentiation of CD16(+) monocytes, or addition of IL-10-containing M14-conditioned medium, significantly enhanced their expression of anti-inflammatory-associated molecules while impairing their acquisition of proinflammatory-related markers. Thus, we propose that M-CSF drives CD14(++)CD16- monocyte differentiation into bona fide anti-inflammatory Mϕs in a self-autonomous manner, whereas M-CSF-treated CD16(+) monocytes generate Mϕs with a skewed proinflammatory profile by virtue of their high activin A expression unless additional anti-inflammatory stimuli such as IL-10 are provided.

Diverging biological roles among human monocyte subsets in the context of tuberculosis infection.

Circulating monocytes (Mo) play an essential role in the host immune response to chronic infections. We previously demonstrated that CD16(pos) Mo were expanded in TB (tuberculosis) patients, correlated with disease severity and were refractory to dendritic cell differentiation. In the present study, we investigated whether human Mo subsets (CD16(neg) and CD16(pos)) differed in their ability to influence the early inflammatory response against Mycobacterium tuberculosis. We first evaluated the capacity of the Mo subsets to migrate and engage a microbicidal response in vitro. Accordingly, CD16(neg) Mo were more prone to migrate in response to different mycobacteria-derived gradients, were more resistant to M. tuberculosis intracellular growth and produced higher reactive oxygen species than their CD16(pos) counterpart. To assess further the functional dichotomy among the human Mo subsets, we carried out an in vivo analysis by adapting a hybrid mouse model (SCID/Beige, where SCID is severe combined immunodeficient) to transfer each Mo subset, track their migratory fate during M. tuberculosis infection, and determine their impact on the host immune response. In M. tuberculosis-infected mice, the adoptively transferred CD16(neg) Mo displayed a higher lung migration index, induced a stronger pulmonary infiltration of murine leucocytes expressing pro- and anti-inflammatory cytokines, and significantly decreased the bacterial burden, in comparison with CD16(pos) Mo. Collectively, our results indicate that human Mo subsets display divergent biological roles in the context of M. tuberculosis infection, a scenario in which CD16(neg) Mo may contribute to the anti-mycobacterial immune response, whereas CD16(pos) Mo might promote microbial resilience, shedding light on a key aspect of the physiopathology of TB disease.

Tolerogenic dendritic cells induce antigen-specific hyporesponsiveness in insulin- and glutamic acid decarboxylase 65-autoreactive T lymphocytes from type 1 diabetic patients.

Tolerogenic dendritic cells (tDC) constitute a promising therapy for autoimmune diseases, since they can anergize T lymphocytes recognizing self-antigens. Patients with type 1 diabetes mellitus (T1D) have autoreactive T cells against pancreatic islet antigens (insulin, glutamic acid decarboxylase 65 -GAD65-). We aimed to determine the ability of tDC derived from T1D patients to inactivate their insulin- and GAD65-reactive T cells. CD14+ monocytes and CD4+CD45RA- effector/memory lymphocytes were isolated from 25 patients. Monocyte-derived DC were generated in the absence (control, cDC) or presence of IL-10 and TGF-ß1 (tDC), and loaded with insulin or GAD65. DC were cultured with T lymphocytes (primary culture), and cell proliferation and cytokine secretion were determined. These lymphocytes were rechallenged with insulin-, GAD65- or candidin-pulsed cDC (secondary culture) to assess whether tDC rendered T cells hyporesponsive to further stimulation. In the primary cultures, tDC induced significant lower lymphocyte proliferation and IL-2 and IFN-γ secretion than cDC; in contrast, tDC induced higher IL-10 production. Lymphocytes from 60% of patients proliferated specifically against insulin or GAD65 (group 1), whereas 40% did not (group 2). Most patients from group 1 had controlled glycemia. The secondary cultures showed tolerance induction to insulin or GAD65 in 14 and 10 patients, respectively. A high percentage of these patients (70-80%) belonged to group 1. Importantly, tDC induced antigen-specific T-cell hyporesponsiveness, since the responses against unrelated antigens were unaffected. These results suggest that tDC therapy against multiple antigens might be useful in a subset of T1D patients.

The Nurr7 agonist Cytosporone B differentially regulates inflammatory responses in human polarized macrophages.

The orphan nuclear receptor Nur77 is involved in diverse cellular processes such as inflammation, proliferation, differentiation and survival. Stimuli like lipopolysaccharide (LPS) and tumor necrosis factor (TNF) increase Nur77 expression in human and murine macrophages, and it has been proposed that Nur77 plays a major role in dampening the inflammatory response. Here, we evaluated the expression and function of Nur77 in human anti-inflammatory and pro-inflammatory macrophages derived from blood monocytes cultured with macrophage colony-stimulating factor (M-MDMs) or granulocyte/macrophage colony-stimulating factor (GM-MDMs), respectively. Nur77 mRNA expression was significantly enhanced in M-MDMs compared with GM-MDMs, both constitutively and upon exposure to Toll-like receptor (TLR)2, 3, and 4 ligands. Nur77 activation with the agonist Cytosporone B (CsnB) significantly suppressed the production of TNF, interleukin (IL)-1ß, IL-6, and IL-8 in GM-MDMs stimulated with LPS. In contrast, it tended to enhance the production of the anti-inflammatory cytokine IL-10. This effect was associated with reduced NF-κB p65 nuclear translocation. Similarly, Nur77 knockdown enhanced TNF production in GM-MDMs. CsnB effectively stimulated the transactivation activity of Nur77 in M-MDMs, but it did not alter cytokine synthesis or p65 nuclear translocation. However, Nur77 seemed to have a role in maintaining the anti-inflammatory profile of M-MDMs, since Nur77-deficient M-MDMs constitutively produced higher levels of TNF transcripts. Thus, in the absence of exogenous agonists, Nur77 activity favors the anti-inflammatory function of M-MDMs, whereas agonistic activation of this receptor preferentially drives attenuation of inflammation in inflammatory macrophages.

Formation of Foamy Macrophages by Tuberculous Pleural Effusions Is Triggered by the Interleukin-10/Signal Transducer and Activator of Transcription 3 Axis through ACAT Upregulation.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its human host relies on numerous immune evasion strategies, such as the deregulation of the lipid metabolism leading to the formation of foamy macrophages (FM). Yet, the specific host factors leading to the foamy phenotype of Mtb-infected macrophages remain unknown. Herein, we aimed to address whether host cytokines contribute to FM formation in the context of Mtb infection. Our approach is based on the use of an acellular fraction of tuberculous pleural effusions (TB-PE) as a physiological source of local factors released during Mtb infection. We found that TB-PE induced FM differentiation as observed by the increase in lipid bodies, intracellular cholesterol, and expression of the scavenger receptor CD36, as well as the enzyme acyl CoA:cholesterol acyl transferase (ACAT). Importantly, interleukin-10 (IL-10) depletion from TB-PE prevented the augmentation of all these parameters. Moreover, we observed a positive correlation between the levels of IL-10 and the number of lipid-laden CD14+ cells among the pleural cells in TB patients, demonstrating that FM differentiation occurs within the pleural environment. Downstream of IL-10 signaling, we noticed that the transcription factor signal transducer and activator of transcription 3 was activated by TB-PE, and its chemical inhibition prevented the accumulation of lipid bodies and ACAT expression in macrophages. In terms of the host immune response, TB-PE-treated macrophages displayed immunosuppressive properties and bore higher bacillary loads. Finally, we confirmed our results using bone marrow-derived macrophage from IL-10-/- mice demonstrating that IL-10 deficiency partially prevented foamy phenotype induction after Mtb lipids exposure. In conclusion, our results evidence a role of IL-10 in promoting the differentiation of FM in the context of Mtb infection, contributing to our understanding of how alterations of the host metabolic factors may favor pathogen persistence.

Cholinergic Modulation of Type 2 Immune Responses.

In recent years, the bidirectional relationship between the nervous and immune system has become increasingly clear, and its role in both homeostasis and inflammation has been well documented over the years. Since the introduction of the cholinergic anti-inflammatory pathway, there has been an increased interest in parasympathetic regulation of both innate and adaptive immune responses, including T helper 2 responses. Increasing evidence has been emerging suggesting a role for the parasympathetic nervous system in the pathophysiology of allergic diseases, including allergic rhinitis, asthma, food allergy, and atopic dermatitis. In this review, we will highlight the role of cholinergic modulation by both nicotinic and muscarinic receptors in several key aspects of the allergic inflammatory response, including barrier function, innate and adaptive immune responses, and effector cells responses. A better understanding of these cholinergic processes mediating key aspects of type 2 immune disorders might lead to novel therapeutic approaches to treat allergic diseases.

Calcium-sensing-receptor (CaSR) controls IL-6 secretion in metastatic breast cancer MDA-MB-231 cells by a dual mechanism revealed by agonist and inverse-agonist modulators.

IL-6 is a tightly controlled pleiotropic cytokine with hormone-like properties whose levels are frequently altered in cancer and inflammatory diseases. In highly invasive MDA-MB-231 breast cancer cells, basal activity of endogenously expressed calcium sensing receptor (CaSR) promotes IL-6 secretion. Interestingly, upon agonist stimulation, CaSR reduces IL-6 levels whereas it promotes secretion of various other cytokines and growth factors, raising intriguing questions about how CaSR signaling modulates IL-6 secretion. Here, using NPS-2143, which acted as an inverse agonist, we show that IL-6 secretion promoted by constitutive activity of CaSR is mechanistically linked to Gαs/PKC, MEK1/2 and mTORC1 signaling pathways, integrated by transactivated EGFR. On the other hand, agonist-stimulated CaSR engages in a Rab11a-dependent trafficking pathway critical to inhibit constitutive IL-6 secretion via the PI3K/AKT and PKC signaling pathways. These results support the emerging potential of CaSR as a therapeutic target in metastatic breast cancer whose pharmacological modulation would reduce IL-6.

CD163L1 and CLEC5A discriminate subsets of human resident and inflammatory macrophages in vivo.

Macrophages (Mϕ) can be differentiated and polarized in vitro from human CD14(+) monocytes under the influence of GM-CSF (GM-Mϕ) and M-CSF (M-Mϕ). GM-Mϕs are proinflammatory and M-Mϕs have an anti-inflammatory phenotype. We found selective expression of the lectin C-type lectin domain family 5 member A (CLEC5A) transcripts in GM-Mϕs and the scavenger receptor CD163 molecule-like 1 (CD163L1) in M-Mϕs by microarray assay. In vitro, CD163L1 expression was induced by IL-10 and M-CSF and CLEC5A by inflammatory cytokines and cell adherence. In secondary lymphoid organs, their respective expression was restricted to CD68(+)/CD163(+) Mϕs that preferentially produced either TNF (CLEC5A(+)) or IL-10 (CD163L1(+)). Mϕs from healthy liver and colon tissue were mostly CD163L1(+), and CLEC5A(+) cells were scarce. In contrast, CLEC5A(+) Mϕs were abundant in the intestinal lamina propria from patients with inflammatory bowel disease (IBD), with higher numbers of CLEC5A(+)CD163L1(+) found compared with those in secondary lymphoid organs. CLEC5A(+) cells were CD14(+)CD209(-)CD11b(+)CD11c(+)TNF(+)IL-10(+), and single positive CD163L1(+) cells were CD14(-)CD209(+)CD11b(-)CD11c(-)TNF(-)IL-10(+) in healthy donors and had lost the ability to produce IL-10 and to express CD209 in those with IBD. In melanomas, CLEC5A(+) tumor-associated Mϕs (TAMs) were not detected in 42% of the cases evaluated, but CD163L1(+) TAMs were found in 100%. Similar to IBD, CD163L1(+) TAMs expressed high levels of CD209 and produced significant amounts of IL-10, and CLEC5A(+) TAMs were CD14(hi) and produced enhanced levels of TNF in metastases. Overall, these results suggest that CD163L1 expression is associated with tissue-resident Mϕs with an anti-inflammatory or anergic phenotype and that CLEC5A(+) Mϕs exhibit TNF-producing ability and might display a proinflammatory effect.

Human CD16+ and CD16- monocyte subsets display unique effector properties in inflammatory conditions in vivo.

Two major subsets of human Mo are identified based on CD14 and CD16 expression: the classical CD16(-) Mo and the minor CD14(+)CD16(+) Mo. In vitro studies suggested distinct function and differentiation potential for each cell population. However, the in vivo relevance of these findings remains unclear. To evaluate the development and function of human Mo in an in vivo model, we transferred both Mo subpopulations into the peritoneum of immunocompromised mice in homeostatic or inflammatory conditions. Inflammation was induced with soluble LPS or particulate zymosan. CD16(+) were more phagocytic and produced higher amounts of TNF and IL-6 than CD16(-) Mo early after transfer with zymosan. They also produced higher levels of ß2-defensin in any condition evaluated, which could represent a new marker for this subpopulation. In contrast, differentiating CD16(-) Mo (24 h after transfer) acquired greater APC capacity in LPS-induced peritonitis, whereas none of the Mo subsets attained this ability with zymosan. CX(3)CL1 supported the survival of both Mo subsets in vivo. Similar Mo subpopulations were present in human peritonitis. These results support the idea of specialized roles of the Mo subset, where CD16(+) might act in an immediate innate immune response, whereas CD16(-) could have a major role as APCs.