A two-step human culture system replicates intestinal monocyte maturation cascade: Conversion of tissue-like inflammatory monocytes into macrophages.

Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte "waterfall" process in vitro by first, generating tissue-like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14+ CD16- monocytes, cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ and IL23, differentiated into CD14+ CD163- cells that displayed a monocyte-like morphology. In vitro generated inflammatory CD14+ CD163- (inflammatory monocyte-like cells) cells promoted IL-1ß-dependent memory Th17 and Th17/Th1 responses, like the CD14+ CD163- mo-like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte-like cells converted to functional CD163+ MΦ following exposure to TGF-ß and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163+ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two-step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.

The Conversion of Human Tissue-Like Inflammatory Monocytes Into Macrophages.

Classical circulating LyC6high murine monocytes differentiate progressively from inflammatory tissue monocytes to mature macrophages (Mϕ) after entry into gut mucosa. This protocol provides a two-step in vitro culture method that replicates the human monocyte maturation cascade. First, purified circulating CD14+ CD16- monocytes exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFNγ), and interleukin 23 (IL-23) differentiate into tissue-like inflammatory monocytes. Next, addition of transforming growth factor beta (TGFß) plus interleukin 10 (IL-10) promotes their maturation into tissue-like Mϕ. Methods to sort these cells after culture are also provided. The fine-tuning of this system might open therapeutic avenues for chronic inflammatory disorders. © 2021 Wiley Periodicals LLC Basic Protocol 1: Isolation of human monocytes from peripheral blood Basic Protocol 2: First step culture for generation of inflammatory monocyte-like cells Basic Protocol 3: Second step culture for differentiation of inflammatory monocyte-like cells into macrophages Alternate Protocol: Sorting and culturing of inflammatory monocyte-like cells.

IL-12 and Mucosal CD14+ Monocyte-Like Cells Induce IL-8 in Colonic Memory CD4+ T Cells of Patients With Ulcerative Colitis but not Crohn's Disease.

BACKGROUND AND AIMS: CD14+ mononuclear phagocytes [MNPs] and T cells infiltrate colon in ulcerative colitis [UC]. Here we investigated how CD14+ MNPs and the cytokines they produce shape the colonic effector T cell profile. METHODS: Colonic or mesenteric lymph node [mLNs] CD4+ T cells isolated from UC or Crohn's disease [CD] patients were stimulated with cytokines or autologous CD14+ MNPs. Cytokine expression was assessed by intracytoplasmic staining and multiplex ELISA. Unsupervised phenotypic multicolour analysis of colonic CD14+ MNPs was performed using the FlowSOM algorithm. RESULTS: Among CD14+CD64+HLA-DR+SIRPα + MNPs, only the pro-inflammatory cytokine-producing CD163- subpopulation accumulated in inflamed UC colon and promoted mucosal IL-1ß-dependent Th17, Th17/Th1, Th17/Th22 but not Th1 responses. Unsupervised phenotypic analysis of CD14+CD64+ MNPs segregated CD163- monocyte-like cells and CD163+ macrophages. Unexpectedly, IL-12, IL-1ß and CD163-, but not CD163+, cells induced IL-8 expression in colonic CD4+ T cells, which co-expressed IFN-γ and/or IL-17 in UC and not CD. The CD163- monocyte-like cells increased the frequency of IL-8+IL-17+/-IFN-γ +/- T cells through IL-1ß and IL-12. Finally, colonic IL-8+ T cells co-expressing GM-CSF, TNF-α and IL-6 were detected ex vivo and, promoted by IL-12 in the mucosa and mLNs in UC only. CONCLUSIONS: Our findings established a link between monocyte-like CD163- MNPs, IL-12, IL-1ß and the detection of colonic memory IL-8-producing CD4+ T cells, which might all contribute to the pathogenesis of UC.

Correction: Two distinct colonic CD14+ subsets characterized by single-cell RNA profiling in Crohn's disease.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptomic Analysis and High-dimensional Phenotypic Mapping of Mononuclear Phagocytes in Mesenteric Lymph Nodes Reveal Differences Between Ulcerative Colitis and Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease [CD] and ulcerative colitis [UC] are distinct forms of inflammatory bowel disease. Heterogeneity of HLA-DR+SIRPα + mononuclear phagocytes [MNPs], including macrophages [MΦ], monocyte-derived [Mono] cells, and dendritic cells [DCs], was reported in gut tissue but not yet investigated in mesenteric lymph nodes [MLNs] of IBD patients. We here compared the phenotype, function, and molecular profile of HLA-DR+SIRPα + MNPs in CD and UC MLNs. METHODS: Cell distribution, morphology, immune function, and transcriptomic [bulk RNAseq] and high-dimensional protein expression profiles [CyTOF] of HLA-DR+SIRPα + MNPs were examined in MLNs of UC [n = 14], CD [n = 35], and non-IBD [n = 12] patients. RESULTS: Elevated frequencies of CD14+CD64+CD163+ [Mono/MΦ-like] MNPs displaying monocyte/MΦ morphology and phagocytic function were a distinct feature of UC MLNs. In CD, the proportion of CD14-CD64-CD163- [DC-like] cells was augmented relative to Mono/MΦ-like cells; DC-like cells drove naïve T cell proliferation, Th1 polarisation, and Th17 TCM plasticity. Gene expression profile corroborated the nature of DC-like cells, best represented by BTLA, SERPINF, IGJ and, of Mono/MΦ-like cells, defined by CD163, MARCO, MAFB, CD300E, S100A9 expression. CyTOF analysis showed that CD123+ plasmacytoid cells predominated over conventional DCs in DC-like cells. Four CD163+ clusters were revealed in Mono/MΦ-like cells, two of which were enriched in MARCO-CD68dimHLA-DRdim monocyte-like cells and MARCOhiCD68hiHLA-DRhi Mɸ, whose proportion increased in UC relative to CD. CONCLUSIONS: Defining the landscape of MNPs in MLNs provided evidence for expansion of CD163+ Mono/MΦ-like cells in UC only, highlighting a distinction between UC and CD, and thus the potential contribution of monocyte-like cells in driving colitis.

Differential Pathogenic Th17 Profile in Mesenteric Lymph Nodes of Crohn's Disease and Ulcerative Colitis Patients.

The drug targets IL23 and IL12 regulate pathogenicity and plasticity of intestinal Th17 cells in Crohn's disease (CD) and ulcerative colitis (UC), the two most common inflammatory bowel diseases (IBD). However, studies examining Th17 dysregulation in mesenteric lymph nodes (mLNs) of these patients are rare. We showed that in mLNs, CD could be distinguished from UC by increased frequencies of CCR6+CXCR3-RORγ+Tbet-CD4+ (Th17) memory T cells enriched in CD62Llow effector memory T cells (TEM), and their differentially expressed molecular profile. Th17 TEM cells (expressing IL17A, IL17F, RORC, and STAT3) displayed a higher pathogenic/cytotoxic (IL23R, IL18RAP, and GZMB, CD160, PRF1) gene signature in CD relative to UC, while non-pathogenic/regulatory genes (IL9, FOXP3, CTLA4) were more elevated in UC. In both CD and UC, IL12 but not IL23, augmented IFNγ expression in Th17 TEM and switched their molecular profile toward an ex-Th17 (Th1*)-biased transcriptomic signature (increased IFNG, and decreased TCF7, IL17A), suggesting that Th17 plasticity occurs in mLNs before their recruitment to inflamed colon. We propose that differences observed between Th17 cell frequencies and their molecular profile in CD and UC might have implications in understanding disease pathogenesis, and thus, therapeutic management of patients with IBD.

Two distinct colonic CD14+ subsets characterized by single-cell RNA profiling in Crohn's disease.

Inflammatory bowel diseases are associated with dysregulated immune responses in the intestinal tissue. Four molecularly identified macrophage subsets control immune homeostasis in healthy gut. However, the specific roles and transcriptomic profiles of the phenotypically heterogeneous CD14+ macrophage-like population in inflamed gut remain to be investigated in Crohn's disease (CD). Here we identified two phenotypically, morphologically and functionally distinct colonic HLADR+SIRPα+CD14+ subpopulations that were further characterized using single-cell RNA-sequencing (scRNAseq) in CD. Frequencies of CD64hiCD163-/dim cells selectively augmented in inflamed colon and correlated with endoscopic score of disease severity. IL-1ß and IL-23-producing CD64hiCD163-/dim cells predominated over TNF-α-producing CD64hiCD163hi cells in lesions. Purified "inflammatory monocyte-like" CD163-, but not "macrophage-like" CD163hi cells, through IL-1ß, promoted Th17/Th1 but not Th1 responses in tissue memory CD4+T cells. Unsupervised scRNAseq analysis that captures the entire HLADR+SIRPα+ population revealed six clusters, two of which were enriched in either CD163- or CD163hi cells, and best defined by TREM1/FCAR/FCN1/IL1RN or CD209/MERTK/MRCI/CD163L1 genes, respectively. Selected newly identified discriminating markers were used beyond CD163 to isolate cells that shared pro-Th17/Th1 function with CD163- cells. In conclusion, a molecularly distinct pro-inflammatory CD14+ subpopulation accumulates in inflamed colon, drives intestinal inflammatory T-cell responses, and thus, might contribute to CD disease severity.

Differential accumulation and function of proinflammatory 6-sulfo LacNAc dendritic cells in lymph node and colon of Crohn's versus ulcerative colitis patients.

Human Slan DCs have been studied in patients with psoriasis, rheumatoid arthritis, cancer, and autoimmune diseases. In this study, we investigated the frequency, phenotype, and function of Slan DCs in blood, colon, as well as mLNs of patients with IBD. We first show that the frequency of circulating CD14(dull)Slan DCs was reduced in CD patients refractory to immunosuppressive drugs or TNF-α blockers relative to untreated CD, UC, and healthy subjects. In blood of CD patients, Slan DCs expressed CD172a, as detected by CD47 fusion protein binding, when compared with its lack of expression in control subjects. Next, we demonstrate that CD172a(+)Slan DCs that produced IL-1ß and TNF-α accumulated in mLNs and colons of CD patients. The CD172a(+)Slan DCs up-regulated their expression of CD14 in CD tissues and the proinflammatory cytokines were produced in CD14(bright)CD172a(+)Slan DCs. By contrast, no difference was noted in the frequency of Slan DCs between inflamed, noninflamed colonic mucosa of UC patients and control, non-IBD donors. Finally, the percentage of cytokine-producing Slan DCs also augmented in response to TLR2 and NOD2 in in vitro stimulation in PBMCs of CD, but not UC, patients. In conclusion, we propose that proinflammatory CD14(bright)CD172a(+)Slan DCs are a distinguishing feature between CD and UC, as these cells accumulate uniquely in mLNs and colonic mucosa of CD patients. Thus, Slan DCs may contribute to CD immunopathogenesis.

Glottal contact quotient in Mediterranean tongue trill.

OBJECTIVE: To determine the effect of a Mediterranean tongue trill (Zalghouta) on estimated glottal closed quotient (CQ). MATERIAL AND METHOD: A total of 10 female subjects participated in this study. Vocal fold CQ was measured for both sustained vowel [a] and the tongue trill named Zlaghouta using electroglottography. Frequencies and means (±standard deviation) were used to describe categorical and continuous variables, respectively. The Wilcoxon signed nonparametric test was used to determine any significant changes in CQ means pretrill and during trill. RESULTS: There was a significant decrease in the mean CQ during the Mediterranean tongue trill (Zalghouta) versus the sustained vowel [a] (P value of 0.002) by 15.98. When stratified by training status there was a significant decrease for the trained group (P value of 0.031) and a nonsignificant decrease for the untrained group (P value of 0.125). CONCLUSION: Zalghouta induces a decrease in the vocal fold contact time. The potential benefits of the Mediterranean tongue trill in vocal therapy need consideration.