Journal: bioRxiv

Article Title: Branched actin networks in innate immune cells mediate host microbiota homeostasis

doi: 10.1101/2024.07.18.604111

Figure Lengend Snippet: (A) The level of Arpc5 and Arpc5l in C5 fl/fl , C5 HetVav or C5 1′Vav bone marrow extracts. ( B ) Lipocalin-2 (LPCN-2) faecal levels in the indicated mice. ( C ) H&E images of intestines (left) together with quantification of the histopathology score (middle) and disease activity index (DAI) (right). ( D ) Immunohistochemistry reveals the increased prevalence of macrophages (top) and neutrophils (bottom) in ileal sections in mice lacking Arpc5. ( E ) Loss of Arpc5 increases the size and cell counts of Mesenteric lymph nodes (mLNs) but not the inguinal lymph node (iLN). ( F ). Mice lacking Arpc5 have increased levels C-reactive protein (CRP) as well as phagocytes in their blood. ( G ) Quantification of the counts of neutrophils (NØs) and Ly6C hi/lo monocytes (MØs) in peripheral blood of the indicated mice. (H) Representative images together with the quantification of the counts of erythroid lineage and immature neutrophil (NØ) in the bone marrow of the indicated mice. ( I ) Quantification of the % of multipotent progenitors (MPP) among the lineage - Sca1 + c-Kit + (LSK) cells. MPP-Ly: lymphoid-biased MPPs; MPP-G/M: myeloid-biased MPPs; MPP-MkE: megakaryocyte and erythroid-biased MPPs; MPP: unbiased MPPs. ( J ) Representative image and weight of spleen in the indicated mice. ( K ) Bacterial colony forming units (CFU) in C5 HetVav or C5 1′Vav mice spleens. ( L ) Schematic of the bone marrow transplantation protocol: CD45.1_wild type mice were lethally irradiated and reconstituted with either CD45.2_controls (WT-Control) or CD45.2_C5 1′Vav (WT-C5 1′Vav ) bone marrow. H&E image of intestines of animals which received wild-type (WT-control) or C5 1′Vav (WT-C5 1′Vav ) bone marrow transplantation. Animals assessed in this figure were 8-15 week old mice n=7 of two independent experiments. Data shown as means ± SEM of at least three independent experiments, n=8. Statistical analysis was performed using Kruskal-Wallis test in B , Mann-Whitney test in C , E, F , H, J, K, L, one-way ANOVA in G and I, or multiple Mann-Whitney test in D . Significant p values are indicated on the graphs. C5 fl/fl (arpc5 fl/fl ), C5 HetVav (arpc5 HetVav ) and C5 1′Vav (arpc5 1′Vav ). ns = not significant.* = p-value < 0.05. ** = p-value < 0.01. *** = p-value < 0.001 and **** = p-value < 0.0001.

Article Snippet: Four week old Arpc5 Cre-Vav1 (C5 1′Vav ) mice received intraperitoneal injection of 5μl/g clodronate_SUV_PEG liposomes (CP-SUV-P-005-005-Liposoma) and 48h later were adoptively transferred with 3 x 10 6 CD115 + isolated from wild-type or C5 1′Vav mice bone marrows following the manufacturer’s instructions (Miltenyi Biotec, #130-096-354).

Techniques: Histopathology, Activity Assay, Immunohistochemistry, Transplantation Assay, Irradiation, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin networks in innate immune cells mediate host microbiota homeostasis

doi: 10.1101/2024.07.18.604111

Figure Lengend Snippet: ( A ) Lipocalin-2 (LPCN-2) faecal levels (left), quantification of the histopathology score (middle) and H&E image of the ileum of 4 weeks old (4wko) C5 HetVav or 4 and 8wko C5 1′Vav animals (right). ( B ) Experimental scheme of antibiotic treatment; 4wko animals were treated with antibiotic cocktail (Atbx) or vehicle (Veh) for 4 weeks and then sampled for analysis. ( C ) Quantification of LPCN-2 levels in faeces (left), and histopathology score (middle) together with H&E image of the ileum in the indicated mice (right). ( D ) Representative immunohistochemistry images and quantification showing Atbx treatment reduces macrophages (top) and neutrophils (bottom) presence in ileal sections in C5 1′Vav mice. (E ) Levels of C-reactive protein (CRP) in the blood of animals with and without antibiotic treatment. (F ) Representative images and weight of spleens animals with and without antibiotics. ( G ) Representative image together with the quantification of numbers of immature neutrophil (NØ) in the bone marrow of mice with and without antibiotic treatment. Data shown as means ± SEM of at least three independent experiments, n=7. Statistical analysis was performed using Kruskal-Wallis tests in A , C , E , F and G, or Mann-Whitney test in D . Significant p values are indicated on the graphs. Controls used were arpc5 HetVav and C5 1′Vav (arpc5 1′Vav ). ns = not significant. * = p-value < 0.05. ** = p-value < 0.01. *** = p-value < 0.001 and **** = p-value < 0.0001.

Article Snippet: Four week old Arpc5 Cre-Vav1 (C5 1′Vav ) mice received intraperitoneal injection of 5μl/g clodronate_SUV_PEG liposomes (CP-SUV-P-005-005-Liposoma) and 48h later were adoptively transferred with 3 x 10 6 CD115 + isolated from wild-type or C5 1′Vav mice bone marrows following the manufacturer’s instructions (Miltenyi Biotec, #130-096-354).

Techniques: Histopathology, Immunohistochemistry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin networks in innate immune cells mediate host microbiota homeostasis

doi: 10.1101/2024.07.18.604111

Figure Lengend Snippet: (A) Levels of C-reactive protein (CRP) in the serum and ( B ) quantification of disease activity index (DAI including values also shown in , left) and histopathology score together with representative H&E images of cecum of control RagKO or C5 1′VavRagKO mice (right). ( C ) Schematic of the bone marrow transplantation protocol: CD45.1_wildtype mice were subjected to lethal irradiation and reconstituted with either CD45.2_control RagKO (WT-CT RagKO ) or CD45.2_C5 1′VavRagKO (WT-C5 1′VavRagKO ) bone marrow. ( D ) Levels of CRP(left) and quantification of the histopathology score in irradiated mice together with representative H&E images of ileum (right). ( E ) Scheme for adoptive cell transfer after busulfan treatment: 4 weeks old C5 1′Vav mice were treated with busulfan and injected with either WT (C5-Rag -/- ) or C5 1′Vav (C5-C5 1′Vav ) innate cells. ( F ) Lipocalin-2 (LPCN-2) faecal levels (left) and quantification of the histopathology score together with representative H&E images of the ileum of busulfan-treated mice (right). ( G ) Clodronate liposome treatment scheme: clodronate liposomes were injected into 4 weeks old animals followed by wild-type (MØ WT ) or C5 1′Vav (MØ KO ) CD115 + cell transplantation n=5 of two independent experiments. ( H ) LPCN-2 faecal levels (left) and quantification of the histopathology score together with representative H&E images of the ileum (right) of indicated animals after clodronate depletion followed by adoptive cell transfer. Data shown as means ± SEM of at least three independent experiments, n=8 except as indicated in (H). Statistical analysis was performed using Mann-Whitney test in A , B (right), D, F and H and multiple Mann-Whitney test in B (left). Significant p values are indicated on the graphs. Controls used were Arpc5 HetVav/RagKO . C5 1′VavRagKO (Arpc5 1′Vav/RagKO ). Clo = clodronate liposomes. ns = not significant. * = p-value < 0.05. ** = p-value < 0.01. *** = p-value < 0.001 and **** = p-value < 0.0001.

Article Snippet: Four week old Arpc5 Cre-Vav1 (C5 1′Vav ) mice received intraperitoneal injection of 5μl/g clodronate_SUV_PEG liposomes (CP-SUV-P-005-005-Liposoma) and 48h later were adoptively transferred with 3 x 10 6 CD115 + isolated from wild-type or C5 1′Vav mice bone marrows following the manufacturer’s instructions (Miltenyi Biotec, #130-096-354).

Techniques: Activity Assay, Histopathology, Control, Transplantation Assay, Irradiation, Injection, Liposomes, MANN-WHITNEY