IL-1β Promotes Staphylococcus aureus Biofilms on Implants in vivo

Implant associated infections represent a serious health burden in clinics since some microorganisms are able to colonize biological surfaces or surfaces of indwelling medical devices and form biofilms. Biofilms represent communities of microorganisms attached to hydrated surfaces and enclosed in self-produced extracellular matrix. This renders them resistant to exogenous assaults like antibiotics or immune effector mechanisms. Little is known regarding the role of the immune system in the formation of biofilms during implant associated infections, largely due to the lack of suitable mouse models. Here we use colonized osmotic pumps in mice to study the interaction of an activated immune system with biofilm-forming Staphylococcus aureus encoding Gaussia luciferase. This approach permits biofilm formation on the osmotic pumps in living animals. It also allows the continuous supply of soluble immune cell activating agents, such as cytokines to study their effect on biofilm formation in vivo. Using non-invasive imaging of the bioluminescent signal emitted by the lux expressing bacteria for quantification of bacterial load in conjunction with light and electron microscopy, we observed that pump-supplied pro-inflammatory cytokine IL-1β strongly increased biofilm formation along with a massive influx of neutrophils adjacent to the biofilm-coated pumps. Thus, our data demonstrate that immune defense mechanisms can augment biofilm formation

A fetal wave of human type 3 effector gamma delta cells with restricted TCR diversity persists into adulthood

Accumulating evidence suggests that the mouse embryonic thymus produces distinct waves of innate effector gamma delta T cells. However, it is unclear whether this process occurs similarly in humans and whether it comprises a dedicated subset of innate-like type 3 effector gamma delta T cells. Here, we present a protocol for high-throughput sequencing of TRG and TRD pairs that comprise the clonal gamma delta TCR. In combination with single-cell RNA sequencing, multiparameter flow cytometry, and TCR sequencing, we reveal a high heterogeneity of gamma delta T cells sorted from neonatal and adult blood that correlated with TCR usage. Immature gamma delta T cell clusters displayed mixed and diverse TCRs, but effector cell types segregated according to the expression of either highly expanded individual V delta 1(+) TCRs or moderately expanded semi-invariant V gamma 9V delta 2(+) TCRs. The V gamma 9V delta 2(+) T cells shared expression of genes that mark innate-like T cells, including ZBTB16 (encoding PLZF), KLRB1, and KLRC1, but consisted of distinct clusters with unrelated V gamma 9V delta 2(+) TCR clones characterized either by TBX21, FCGR3A, and cytotoxicity-associated gene expression (type 1) or by CCR6, RORC, IL23R, and DPP4 expression (type 3). Effector gamma delta T cells with type 1 and type 3 innate T cell signatures were detected in a public dataset of early embryonic thymus organogenesis. Together, this study suggests that functionally distinct waves of human innate-like effector gamma delta T cells with semi-invariant V gamma 9V delta 2(+) TCR develop in the early fetal thymus and persist into adulthood

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca2+ sensor GCaMP6S to visualize in real-time chemokine-induced Ca2+ signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo

IL-1β Promotes Biofilms on Implants .

Implant associated infections represent a serious health burden in clinics since some microorganisms are able to colonize biological surfaces or surfaces of indwelling medical devices and form biofilms. Biofilms represent communities of microorganisms attached to hydrated surfaces and enclosed in self-produced extracellular matrix. This renders them resistant to exogenous assaults like antibiotics or immune effector mechanisms. Little is known regarding the role of the immune system in the formation of biofilms during implant associated infections, largely due to the lack of suitable mouse models. Here we use colonized osmotic pumps in mice to study the interaction of an activated immune system with biofilm-forming Staphylococcus aureus encoding Gaussia luciferase. This approach permits biofilm formation on the osmotic pumps in living animals. It also allows the continuous supply of soluble immune cell activating agents, such as cytokines to study their effect on biofilm formation in vivo. Using non-invasive imaging of the bioluminescent signal emitted by the lux expressing bacteria for quantification of bacterial load in conjunction with light and electron microscopy, we observed that pump-supplied pro-inflammatory cytokine IL-1β strongly increased biofilm formation along with a massive influx of neutrophils adjacent to the biofilm-coated pumps. Thus, our data demonstrate that immune defense mechanisms can augment biofilm formation

Shift of graft-versus-host-disease target organ tropism by dietary vitamin A.

Gut-homing of donor T cells is causative for the development of intestinal GvHD in recipients of allogeneic hematopoietic stem cell transplantation (HSCT). Expression of the gut-specific homing receptors integrin-α4β7 and chemokine receptor CCR9 on T cells is imprinted in gut-associated lymphoid tissues (GALT) under the influence of the vitamin A metabolite retinoic acid. Here we addressed the role of vitamin A deficiency in HSCT-recipients for donor T cell migration in the course of experimental GvHD. Vitamin A-deficient (VAD) mice were prepared by feeding them a vitamin A-depleted diet. Experiments were performed in a C57BL/6 into BALB/c model of acute GvHD. We found that expression of integrin-α4β7 and CCR9 in GALT was reduced in VAD recipients after HSCT. Competitive in vivo homing assays showed that allogeneic T cells primed in VAD mice did not home as efficiently to the intestine as T cells primed in mice fed with standard diet (STD). The course of GvHD was ameliorated in VAD HSCT-recipients and, consequently, their survival was prolonged compared to recipients receiving STD. However, VAD-recipients were not protected and died of clinical GvHD. We found reduced numbers of donor T cells in the intestine but increased cell counts and tissue damage in other organs of VAD-recipients. Furthermore, we observed high IFN-γ(+)CD4(+) and low FoxP3(+)CD4(+) frequencies of total donor CD4(+) T cells in VAD as compared to STD recipients. Taken together, these results indicate that dietary vitamin A deficiency in HSCT-recipients changed target organ tropism in GvHD but also resulted in fatal inflammation after HSCT

Manifold Roles of CCR7 and Its Ligands in the Induction and Maintenance of Bronchus-Associated Lymphoid Tissue

Summary: The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects of dendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation. : Fleige et al. demonstrate that CCR7 and its ligands CCL19, CCL21-serine, and CCL21-leucine orchestrate multiple steps during induction and maintenance of bronchus-associated lymphoid tissue (BALT) including DC-based initial developmental processes as well as homing of blood-derived lymphocytes via HEVs to established BALT. Keywords: BALT, CCR7, CCL21, DCs, MVA, plt/plt, HEV

Reduced accumulation of donor T cells in the intestine and increased accumulation of donor T cells in the liver of VAD recipients during acute GvHD.

<p>Mice were analyzed for donor T cell (Thy1.1) occurrence in SPL, mLN, liver and small intestine (SI) at day 21 after transplantation. (A) Each dot represents the percentage of infiltrating Thy1.1⁺CD4⁺ T cells of all Thy1.1⁺CD4⁺ T cells. (B) Each dot represent the percentage of infiltrating Thy1.1⁺CD8⁺ T cells of all Thy1.1⁺CD8⁺ T cells. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (C) Each bar represents absolute numbers of infiltrating Thy1.1⁺CD4⁺ T cells from STD or VAD recipients. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (D) Each bar represents absolute numbers of infiltrating Thy1.1⁺CD4⁺ T cells from STD or VAD recipients. Bars indicate mean values. N = 4–5/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments.</p

Vitamin A deficiency of recipient mice leads to increased Th1 cells and decreased FoxP3⁺ Treg cells during GvHD.

<p>STD or VAD recipient mice were analyzed for CD4⁺ T cell polarization status in SPL, mLN, liver and small intestine (SI) at day 21 after transplantation. (<b>A</b>) Each bar represents the percentage of FoxP3⁺ CD4⁺ T cells of CD4⁺ T cells isolated from the indicated organ. (<b>B</b>) Each bar represents the percentage of IFN-γ⁺ CD4⁺ T cells of CD4⁺ T cells isolated from the indicated organ.</p

Vitamin A deficiency led to increased hepatic inflammation.

<p>(<b>A</b>) Serum cytokine levels were analyzed using the Cytometric Bead Array, Mouse Inflammation Kit (BD Biosciences). N = 5–6/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (<b>B</b>) Expression analyses of cytokine levels of GvHD target organs using RT-PCR (N = 6/group) Data are combined from two independent experiments (<b>C</b>) Macroscopic comparison of spleen sizes of VAD and STD recipient mice. N = 3/group. One of two representative experiments is shown. (<b>D</b>) Liver enzymes in the course of GvHD in VAD and STD recipients. N = 5–6/group. **: p<0.01; ***: p<0.001. Data are combined from two independent experiments. (<b>E, F</b>) Representative sections from formalin fixed, paraffin embedded livers and small intestines harvested at 21 days post transplantation. The 4–6 µm slides were stained with hematoxylin and eosin.</p

Vitamin A deficiency in HSCT recipients prolongs survival but does not protect from lethal GvHD.

<p>After lethal irradiation, STD BALB/c or VAD BALB/c recipients received 5×10⁶ C57BL/6 T cell depleted bone marrow cells supplemented with 1×10⁶ T cells (N = 14/group). STD BALB/c or VAD BALB/c recipients that received syngeneic (BALB/c) grafts were used as controls (N = 4/group). Data are combined from two independent experiments.</p