CXCR4 is dispensable for T cell egress from chronically inflamed skin via the afferent lymph.

T cell recirculation through extralymphoid tissues is essential to immune surveillance, host defense and inflammation. In this process, T cells enter the tissue from the blood and subsequently leave via the afferent lymph. In the absence of inflammation, T cells require CCR7 expression to egress from the skin or lung, which is consistent with the constitutive expression of the CCR7 ligand CCL21 on lymphatic endothelium. However, during chronic inflammation alternative chemoattractants come into play, allowing Ccr7-deficient (Ccr7-/-) T cells to egress efficiently from affected skin. As T cell egress from inflamed sites is a potential control point of the inflammatory response, we aimed to determine alternative T cell exit receptors using a mouse and a sheep model. We show that CCR7+ and CCR7- T cells exiting from the chronically inflamed skin were highly responsive to the CXCR4 ligand CXCL12, which was induced in the lymphatics in the inflamed site. Based on these findings, we hypothesized that CXCR4 mediates T cell egress from inflamed skin. However, pharmacological inhibition of CXCR4 did not affect the tissue egress of wildtype or Ccr7-/- CD4 and CD8 T cells after adoptive transfer into chronically inflamed skin. Similarly, adoptively transferred Cxcr4-/- Ccr7-/- and Ccr7-/- T cells egressed from the inflamed skin equally well. Based on these data, we conclude that, while CXCR4 might play an essential role for other cell types that enter the afferent lymphatics, it is dispensable for T cell egress from the chronically inflamed skin

Cxcr4 deficiency does not reduce the tissue exit of Ccr7^−/− T cells from chronically inflamed skin.

<p>(<b>A–C</b>) Adoptive transfer exit experiments with fetal liver chimeric mice as a source of donor splenocytes. As depicted in the experimental scheme (<b>A</b>), fetal liver chimeras were generated by reconstituting lethally irradiated CD45.1⁺ wildtype recipient mice with fetal liver cells from (CD45.2⁺) <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− or (CD45.2⁺) <i>Ccr7</i>^−/− or (CD45.1⁺) wildtype donors. Splenocytes from the fetal liver chimeras were labeled with eFlour670 and CFSE, so that the combination of congenic maker and fluorescent label uniquely marked cells of each genotype. Labeled splenocytes of all three genotypes were mixed and co-injected into chronically inflamed footpads. (<b>B</b>) Analysis of migrated lymphocyte subsets recovered in the draining popliteal lymph nodes 12 h post cell transfer. Data is presented as the ratio of migrated to input cells for <i>Ccr7</i>^−/− or <i>Cxcr4</i>^−/−<i>Ccr</i>7^−/− relative to wildtype cells of each subset. Connected lines represent individually analyzed recipient mice. Horizontal, non-connecting lines indicate the mean of each group. (<b>C</b>) Flow cytometric analysis of memory (CD45RB^l°) versus naïve (CD45RB^hi) phenotype CD4 and CD8 T cells in injected and migrated populations for each genotype. Data combined from 2 experiments analyzing 5–10 recipient mice per experiment (<b>B</b>) or the cell phenotype of one out two experiment with similar results (<b>C</b>) is shown. WT, wildtype.</p

Afferent lymph vessels in chronically inflamed skin express CXCL12.

<p>Immunofluorescence staining of frozen sections of chronically inflamed skin (21 days after inflammation elicited by subcutaneous injection of CFA) for CXC12 and CCL21 by LYVE-1⁺ lymph vessels using DAPI counterstaining. One representative image of lymph vessels analyzed in 5 mice is shown. Scale bars, 10 µm.</p

Ovine T cells that exit the inflamed and uninflamed skin efficiently migrate to CXCL12.

<p>Lymphocytes traveling in afferent lymph draining uninflamed or chronically inflamed skin (>d15 post induction of inflammation with CFA) were collected from sheep, and chemotaxis was tested toward mouse CCL21 and human CXCL12 in a Transwell chemotaxis assay. Migration of CD4, CD8 and γδ T cells is expressed as the percentage of each T cell subset that migrated to the lower chamber. Data points represent the mean migration of T cells from individual animals based on triplicate wells for each concentration, and the mean ± SEM for all animals analyzed is shown. N = 5–7 animals per condition and T cell subset.</p

Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7^−/− T cells.

<p>(<b>A–B</b>) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 (<b>A</b>) and CCL21 (<b>B</b>) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. (<b>C–E</b>) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled <i>Ccr7</i>^−/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype (<b>C</b>) and <i>Ccr7</i>^−/− (<b>D</b>) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results (<b>C</b> and <b>D</b>) or both experiments combined (<b>E</b>) are shown. WT, wildtype.</p

Timed Action of IL-27 Protects from Immunopathology while Preserving Defense in Influenza

Infection with influenza virus can result in massive pulmonary infiltration and potentially fatal immunopathology. Understanding the endogenous mechanisms that control immunopathology could provide a key to novel adjunct therapies for this disease. Here we show that the cytokine IL-27 plays a crucial role in protection from exaggerated inflammation during influenza virus infection. Using Il-27ra−/− mice, IL-27 was found to limit immunopathology, neutrophil accumulation, and dampened TH1 or TH17 responses via IL-10–dependent and -independent pathways. Accordingly, the absence of IL-27 signals resulted in a more severe disease course and in diminished survival without impacting viral loads. Consistent with the delayed expression of endogenous Il-27p28 during influenza, systemic treatment with recombinant IL-27 starting at the peak of virus load resulted in a major amelioration of lung pathology, strongly reduced leukocyte infiltration and improved survival without affecting viral clearance. In contrast, early application of IL-27 impaired virus clearance and worsened disease. These findings demonstrate the importance of IL-27 for the physiological control of immunopathology and the potential value of well-timed IL-27 application to treat life-threatening inflammation during lung infection

IL-27 directly modulates IFN-γ production by T cells and indirectly regulates IL-17 response via IL-10.

<p><i>Il-27ra^−/−</i> and C57BL/6 mice were infected with 2500 EID influenza virus. At 9 d.p.i, IL-10 levels in the (<b>A</b>) BAL fluid or (<b>B</b>) supernatants of enriched lymphocytes after PMA/ionomycin restimulation were determined by ELISA. (<b>C</b>) Total numbers of IL-10⁺ T cells in the BAL were analyzed by FACS after PMA/ionomycin restimulation. Representative FACS plots shown are gated CD4⁺ T cells from BAL. (<b>D</b>) <i>Il-10^−/−</i> or C57BL/6 (WT) mice were infected with a sublethal dose influenza virus. At 7 d.p.i., influenza virus peptide-specific IFN-γ or IL-17-producing CD4⁺ T cells in the respiratory tract were assessed by FACS. (<b>E and F</b>) Naive CD8⁺ T cells were activated with plate-bound anti-CD3 and anti-CD28 in Tc1 polarizing conditions in the presence or absence of rIL-27 and/or anti-IL-10 receptor blocking antibody (αIL-10R). After 3 days (d), cells were transferred to plates, then media was replenished with rIL-2, rIL-27 and/or αIL-10R antibody for additional 2 d, for a total of 5 d in culture. IFN-γ or IL-2 production by Tc1 cells were analyzed by FACS. <b>A</b> to <b>D</b> were pooled from two independent experiments with similar results. <b>E</b> and <b>F</b> represent data from three independent experiments with similar results. <i>P</i> values were determined by unpaired two-tailed Student's <i>t</i> test. Values are means ± s.d. ns, not significant.</p

rIL-27 treatment at an early stage of infection aggravates disease severity and impairs viral clearance.

<p>C57BL/6 mice were challenged with a sublethal dose influenza virus then treated daily with rIL-27 from 1–7 d.p.i. Non-treated control mice (NT) were injected with PBS. (<b>A</b>) Weight loss of rIL-27 treated (<i>n</i> = 5) or NT (<i>n</i> = 5) mice. Arrows (↓) indicate time of treatment. At 7 d.p.i., (<b>B</b>) histological scores of H&E-stained lungs from treated or NT groups and (<b>C</b>) viral <i>pa</i> mRNA expression were determined. (<b>D</b>) Numbers of influenza virus peptide-specific IL-17 or IFN-γ-producing T cells, (<b>E</b>) influx of neutrophils, (<b>F</b>) monocytes and NK cells in the lungs at 9 d.p.i. were determined by FACS. Gated cells in FACS plots in <b>e</b> indicate the percentage of neutrophils from total lung cells. Data from <b>A</b> are representative of two independent treatment experiments. Data from <b>B</b> to <b>F</b> are pooled from at least two independent experiments. <i>P</i> values were determined by unpaired two-tailed Student's <i>t</i> test. Values are means ± s.d. except for A, s.e.m.; ns, not significant.</p