Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis

Immunity.

2018 Nov 13

Girbl T, Lenn T, Perez L, Rolas L, Barkaway A, Thiriot A, del Fresno C, Lynam E, Hub E, Thelen M, Graham G, Alon R, Sancho D, von Andrian UH, Voisin MB, Rot A, Nourshargh S.
PMID: 30446388 | DOI: 10.1016/j.immuni.2018.09.018

Neutrophils require directional cues to navigate through the complex structure of venular walls and into inflamed tissues. Here we applied confocal intravital microscopy to analyze neutrophil emigration in cytokine-stimulated mouse cremaster muscles. We identified differential and non-redundant roles for the chemokines CXCL1 and CXCL2, governed by their distinct cellular sources. CXCL1 was produced mainly by TNF-stimulated endothelial cells (ECs) and pericytes and supported luminal and sub-EC neutrophil crawling. Conversely, neutrophils were the main producers of CXCL2, and this chemokine was critical for correct breaching of endothelial junctions. This pro-migratory activity of CXCL2 depended on the atypical chemokine receptor 1 (ACKR1), which is enriched within endothelial junctions. Transmigrating neutrophils promoted a self-guided migration response through EC junctions, creating a junctional chemokine "depot" in the form of ACKR1-presented CXCL2 that enabled efficient unidirectional luminal-to-abluminal migration. Thus, CXCL1 and CXCL2 act in a sequential manner to guide neutrophils through venular walls as governed by their distinct cellular sources.

CD11b suppresses TLR activation of nonclassical monocytes to reduce primary graft dysfunction after lung transplantation

The Journal of clinical investigation

2022 Jul 15

Querrey, M;Chiu, S;Lecuona, E;Wu, Q;Sun, H;Anderson, M;Kelly, M;Ravi, S;Misharin, AV;Kreisel, D;Bharat, A;Budinger, GRS;
PMID: 35838047 | DOI: 10.1172/JCI157262

Primary graft dysfunction (PGD) is the leading cause of postoperative mortality in lung transplant recipients and the most important risk factor for development of chronic lung allograft dysfunction. The mechanistic basis for the variability in the incidence and severity of PGD between lung transplant recipients is not known. Using a murine orthotopic vascularized lung transplant model, we found that redundant activation of Toll-like receptors 2 and 4 (TLR2 and -4) on nonclassical monocytes activates MyD88, inducing the release of the neutrophil attractant chemokine CXCL2. Deletion of Itgam (encodes CD11b) in nonclassical monocytes enhanced their production of CXCL2 and worsened PGD, while a CD11b agonist, leukadherin-1, administered only to the donor lung prior to lung transplantation, abrogated CXCL2 production and PGD. The damage-associated molecular pattern molecule HMGB1 was increased in peripheral blood samples from patients undergoing lung transplantation after reperfusion and induced CXCL2 production in nonclassical monocytes via TLR4/MyD88. An inhibitor of HMGB1 administered to the donor and recipient prior to lung transplantation attenuated PGD. Our findings suggest that CD11b acts as a molecular brake to prevent neutrophil recruitment by nonclassical monocytes following lung transplantation, revealing an attractive therapeutic target in the donor lung to prevent PGD in lung transplant recipients.

Emergence of highly profibrotic and proinflammatory Lrat+ Fbln2+ HSC subpopulation in alcoholic hepatitis

Hepatology (Baltimore, Md.)

2022 Oct 01

Balog, S;Fujiwara, R;Pan, SQ;El-Baradie, KB;Choi, HY;Sinha, S;Yang, Q;Asahina, K;Chen, Y;Li, M;Salomon, M;Ng, SW;Tsukamoto, H;
PMID: 36181700 | DOI: 10.1002/hep.32793

Relative roles of HSCs and portal fibroblasts in alcoholic hepatitis (AH) are unknown. We aimed to identify subpopulations of collagen type 1 alpha 1 (Col1a1)-expressing cells in a mouse AH model by single-cell RNA sequencing (scRNA-seq) and filtering the cells with the HSC (lecithin retinol acyltransferase [Lrat]) and portal fibroblast (Thy-1 cell surface antigen [Thy1] and fibulin 2 [Fbln2]) markers and vitamin A (VitA) storage.Col1a1-green fluorescent protein (GFP) mice underwent AH, CCl4 , and bile duct ligation (BDL) procedures to have comparable F1-F2 liver fibrosis. Col1a1-expressing cells were sorted via FACS by VitA autofluorescence and GFP for single-cell RNA sequencing. In AH, approximately 80% of Lrat+Thy1-Fbln2- activated HSCs were VitA-depleted (vs. ~13% in BDL and CCl4 ). Supervised clustering identified a subset co-expressing Lrat and Fbln2 (Lrat+Fbln2+), which expanded 44-fold, 17-fold, and 1.3-fold in AH, BDL, and CCl4 . Lrat+Fbln2+ cells had 3-15-times inductions of profibrotic, myofibroblastic, and immunoregulatory genes versus Lrat+Fbln2- cells, but 2-4-times repressed HSC-selective genes. AH activated HSCs had up-regulated inflammatory (chemokine [C-X-C motif] ligand 2 [Cxcl2], chemokine [C-C motif] ligand 2), antimicrobial (Il-33, Zc3h12a), and antigen presentation (H2-Q6, H2-T23) genes versus BDL and CCl4 . Computational deconvolution of AH versus normal human bulk-liver RNA-sequencing data supported an expansion of LRAT+FBLN2+ cells in AH; AH patient liver immunohistochemistry showed FBLN2 staining along fibrotic septa enriched with LRAT+ cells; and in situ hybridization confirmed co-expression of FBLN2 with CXCL2 and/or human leukocyte antigen E in patient AH. Finally, HSC tracing in Lrat-Cre;Rosa26mTmG mice detected GFP+FBLN2+ cells in AH.A highly profibrotic, inflammatory, and immunoregulatory Lrat+Fbln2+ subpopulation emerges from HSCs in AH and may contribute to the inflammatory and immunoreactive nature of AH.

Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection

Immunity

2022 Jul 15

Dong, X;Limjunyawong, N;Sypek, EI;Wang, G;Ortines, RV;Youn, C;Alphonse, MP;Dikeman, D;Wang, Y;Lay, M;Kothari, R;Vasavda, C;Pundir, P;Goff, L;Miller, LS;Lu, W;Garza, LA;Kim, BS;Archer, NK;Dong, X;
PMID: 35882236 | DOI: 10.1016/j.immuni.2022.06.021

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

The proton-activated receptor GPR4 modulates intestinal inflammation.

J Crohns Colitis.

2017 Nov 09

Wang Y, de Vallière C, Imenez Silva PH, Leonardi I, Gruber S, Gerstgrasser A, Melham H, Weber A, Leucht K, Wolfram L, Hausmann M, Krieg C, Thomasson K, Boyman O, Frey-Wagner I, Rogler G, Wagner CA.
PMID: 29136128 | DOI: 10.1093/ecco-jcc/jjx147

Abstract

BACKGROUND AND AIMS:

During active inflammation tissue intraluminal intestinal pH is decreased in patients with inflammatory bowel disease (IBD). Acidic pH may play a role in IBD pathophysiology. Recently, proton sensing G-protein coupled receptors were identified, including GPR4, OGR1 (GPR68), and TDAG8 (GPR65). We investigated whether GPR4 is involved in intestinal inflammation.

METHODS:

The role of GPR4 was assessed in murine colitis models: chronic dextran sulphate sodium (DSS) administration and by crossbreeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase (MPO) activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes (LPLs) were analyzed.

RESULTS:

Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/- /Il-10-/- double knock-outs the onset and progression of rectal prolapse were significantly delayed and mitigated compared to Gpr4+/+ /Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4 + T-helper cell infiltration, IFN-γ, iNOS, MCP-1 (CCL2), CXCL1 and CXCL2 expression compared to controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages.

CONCLUSION:

Absence of GPR4 ameliorates colitis in IBD animal models indicating an important regulatory rolein mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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