Mutagenesis and functional Characterization of the human CCR3 Receptor

Asthma is a chronic-inflammatory disease of the airways and about 15% of the population in the industrialized countries suffer from it. The characteristic inflammation of the lung is, amongst others, caused by eosinophilic granulozytes which get activated via chemotactic receptor 3 (CCR3). In order to stop activation of eosinophils and the following inflammation and destruction of the lung tissue, research aims to develop specific receptor antagonists. This turns CCR3 into an interesting and promising therapeutic target. To better understand the interaction of receptor and ligands a receptor model was established which should be verified experimentally. Eight specific amino acids (AA) which are located in the transmembranal region (TMR) of CCR3 were mutated and CHO-cells were transfected with them. The influence of the mutations on ligand-receptor interaction were investigated using an receptor internalisation assay. The data obtained clearly demonstrate that some AA (Y113, H114, Y291) have strong influence on the interaction of ligands with CCR3. For other AA (R95, Y41) it could be shown that they either decrease or even increase interaction of different ligands. Both could be demonstrated for natural ligands as well as agonistic and antagonistic small molecules. This was very surprising as up to now it is believed that natural ligands only interact with the N-terminal region of the receptor, but not with the TMR.Based on the data obtained some of the investigated AA are clearly involved in ligand-receptor interaction and/or signal transduction. Comparisons to similar GPCR studies also suggest an impairment of binding of the ligands and therefore overlapping interaction sides of natural ligands and small molecules

Mouse Model of Reversible Intestinal Inflammation.

Current therapies to treat inflammatory bowel disease by dampening excessive inflammatory immune responses have had limited success ( Reinisch et al., 2011 ; Rutgeerts et al., 2005 ; Sandborn et al., 2012 ). To develop new therapeutic interventions, there is a need for better understanding of the mechanisms that are operative during mucosal healing (Pineton de Chambrun et al., 2010 ). To this end, a reversible model of colitis was developed in which colitis induced by adoptive transfer of naïve CD4+ CD45RBhi T cells in lymphopenic mice can be reversed through depletion of colitogenic CD4+ T cells ( Brasseit et al., 2016 )

IFN-γ induces MHCII expression on IECs.

<p>(A) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ intestinal epithelial cells (IEC) isolated from Rag1−/− or Rag1−/− IFN-γ−/− mice that were adoptively transferred with CD4⁺ CD45RB^hi T cells from WT or IFN-γ−/− mice shown as means and SEM in representative histograms (<i>n</i> = 3 mice per group). (B–C) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs (B) and CD4⁺ T cells from the colonic intestinal epithelium (C) isolated from <i>H. hepaticus</i>-infected, anti-IL-10R mAb-administered pIV−/− K14 CIITA Tg, pIV+/− K14 CIITA Tg or pIV+/− K14 CIITA Tg that were treated with neutralizing anti-IFN-γ mAb. Shown are representative FACS plots, means and SEM from two pooled experiments (<i>n</i> = 4–7 mice per group). αIFN-y, anti-interferon-γ monoclonal antibodies; FSC, forward scatter; IFN, interferon; Rag, recombination activating gene; WT, wild type;</p

Colitic pIV−/− K14 CIITA Tg mice lack inducible MHCII expression by colonic IECs.

<p>(A–B) Frequency of CD45.2⁻ EpCAM⁺ MHCII⁺ IECs isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms (A) and summarized data as mean (B) from three pooled experiments (<i>n = </i>8–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IEC, intestinal epithelial cell;</p

Treating murine inflammatory diseases with an anti-erythrocyte antibody

Treatment of autoimmune and inflammatory diseases typically involves immune suppression. In an opposite strategy, we show that administration of the highly inflammatory erythrocyte-specific antibody Ter119 into mice remodels the monocyte cellular landscape, leading to resolution of inflammatory disease. Ter119 with intact Fc function was unexpectedly therapeutic in the K/BxN serum transfer model of arthritis. Similarly, it rapidly reversed clinical disease progression in collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis and completely corrected CAIA-induced increase in monocyte Fcγ receptor II/III expression. Ter119 dose-dependently induced plasma chemokines CCL2, CCL5, CXCL9, CXCL10, and CCL11 with corresponding alterations in monocyte percentages in the blood and liver within 24 hours. Ter119 attenuated chemokine production from the synovial fluid and prevented the accumulation of inflammatory cells and complement components in the synovium. Ter119 could also accelerate the resolution of hypothermia and pulmonary edema in an acute lung injury model. We conclude that this inflammatory anti-erythrocyte antibody simultaneously triggers a highly efficient anti-inflammatory effect with broad therapeutic potential

Chronic <i>H. hepaticus</i> infection plus anti-IL10R mAb treatment induces colitis in pIV−/− K14 CIITA Tg mice.

<p>(A) Development of body weight during anti-IL-10R mAb or isotype treatment of <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls (<i>n = </i>9–11 per group). (B) Serum albumin concentrations in feces collected on days 26–30 (<i>n = </i>6–8 per group). Data are shown as mean and s.d. and represent two pooled experiments. (C–D) Colon histopathological analysis on day 32; (C) colitis scores, data displayed as mean, and (D) representative photomicrographs of colon sections, stained with hematoxylin and eosin. Bar, 100 µm. Data represent three pooled experiments (<i>n = </i>9–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies;</p

Innate effector cells and proinflammatory cytokines are elevated in colitic pIV−/− K14 CIITA Tg mice.

<p>(A–B) Frequency of Ly6G⁺ neutrophil granulocytes (A) and CD11b⁺ Ly6C⁺ inflammatory monocytes (B) isolated from the colonic intestinal epithelium (cIE, left panel) and the colonic lamina propria (cLP, right panel) of <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. (C) <i>ccl3</i>, <i>ccl4</i>, <i>ccl5</i>, <i>il1b</i> and <i>il6</i> mRNA expression levels in colon explants. (D) IL-1β, TNF-α, IL-12p40, CXCL9 and VEGF secretion upon <i>ex vivo</i> organ culture of colon explants. All data represent three pooled experiments (<i>n = </i>9–11 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; IL, interleukin; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor;</p

Colitic pIV−/− K14 CIITA Tg mice display elevated Th1 cells, IFN-γ, and CD4⁺ T cell: FoxP3⁺ Treg cell ratios.

<p>(A–B) Frequency of CD3⁺ CD4⁺ and CD8⁺ T cells isolated from anti-IL-10R mAb or isotype treated, <i>H. hepaticus</i>-infected pIV−/− K14 CIITA Tg mice or pIV+/− K14 CIITA Tg controls. Representative histograms from the colonic intestinal epithelium (cIE) (A) and summarized data (B) from cIE (left) and the colonic lamina propria (cLP) (right) as mean. (C) <i>ifng</i> and <i>tbet</i> mRNA expression levels in colon explants. (D) IFN-γ secretion upon <i>ex vivo</i> organ culture of colon explants as means and s.d. (A–D) Data represent three pooled experiments (<i>n = </i>9–11 per group). (E) Ratio of absolute numbers of CD4⁺ T cells:absolute numbers of CD25⁺ FoxP3⁺ Treg cells from cIE (left) and cLP (right) as mean from two pooled experiments (<i>n = </i>7–10 per group). αIL10R, anti-interleukin-10 receptor monoclonal antibodies; FoxP3, forkhead box P3; IFN, interferon;</p

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD