Fibroblast activation protein is dispensable in the anti-influenza immune response in mice

<div><p>Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4⁺ and CD8⁺ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity.</p></div

The pathologic response of FAP knockout (gko) versus wildtype (WT) mice to intranasal influenza infection and the associated changes in expression of DPP4 family members.

<p>FAP knockout (gko; closed symbols) and WT (open symbols) mice were intranasally infected with 50 pfu PR/8 influenza virus. <b>A.</b> Real-time quantitative PCR results showing relative expression of FAP normalised to the housekeeping gene <i>hprt</i> in the mediastinal lymph nodes (mdLN), and normalised to the <i>18S RNA</i> gene in the lungs (bottom) of uninfected (-) and infected (+) WT mice on day 7 post-infection. Statistical significance was tested using Mann-Whitney test. <b>B.</b> Weight loss response in influenza-infected 10-week old female FAP knockout (closed symbols) and WT (open symbols) mice (n = 7). Results from one of three replicate experiments are shown. Statistical significance was tested using Student’s t-test. * p< 0.05. <b>C.</b> Real-time quantitative PCR results showing relative expression of influenza virus (Inf A), normalised to the 18S RNA gene in the lungs of infected FAP knockout (closed symbols) and WT (open symbols) mice on day 7 post-infection (n = 5 per group). Inf A expression was not detected in uninfected lungs. Each symbol represents data from one mouse and the bar represents the mean±SEM. Statistical significance was tested using the Mann-Whitney test. <b>D.</b> Expression of DPP4 family members DPP4, DPP8 and DPP9 in uninfected and infected FAP knockout (closed symbols) and WT (open symbols) mice. Each symbol represents data from one mouse and the bar represents the mean±SEM. Results from one of two experiments are shown. Statistical significance was tested using the Mann-Whitney test.</p

Effects of genetic deficiency of FAP on the numbers and phenotype of leukocytes at steady state.

<p>Organs were harvested from FAP knockout (gko; closed symbols) and wildtype (WT; open symbols) C57BL/6 mice. <b>A.</b> The numbers of thymic CD4^-CD8^- double negative (DN), CD4⁺CD8⁺ double positive (DP) CD4⁺CD8^- and CD8⁺CD4^- single positive (SP) populations. <b>B.</b> The numbers of splenic CD4⁺ and CD8⁺ T cells. <b>C.</b> The percentages of CD44^- naïve, CD25⁺ regulatory and CD44^hi activated/ memory CD4⁺ T cells in the thymus and the spleen. <b>D.</b> The percentages of CD8⁺ T cell subsets gated according to their expression of CD44 and CD62L in the thymus and the spleen. <b>E.</b> The numbers of dendritic cells (DC) in the spleen, peripheral LN (pLN), mesenteric LN (mLN) and thymus in FAP knockout and WT mice at steady state. <b>F.</b> The geometric mean fluorescence index (MFI) of surface expression of CD86 (top) and B7H1/PD-L1 (bottom) on DC in the spleen, pLN, mLN and thymus. <b>G.</b> The numbers of splenic CD11b⁺Ly6G⁺ neutrophils. <b>H.</b> The numbers of splenic CD19⁺ B cells. Each symbol represents one mouse and the bar represents the mean. Except for part <b>F</b>, results were pooled from two independent experiments of n = 4 mice each. For <b>F</b>, results from one of three experiments (n = 4 mice each) are shown. Statistical significance was tested using the Mann–Whitney test. * p<0.05, ** p<0.01.</p

T and B cell responses to influenza in FAP knockout (gko) and wildtype (WT) mice.

<p><b>A & B.</b> FAP knockout (gko; closed symbols) and wildtype (WT; open symbols) mice received intravenous transfer of donor OT-1 cells and on the next day were intranasally inoculated with 100 pfu PR/8-OVA influenza virus. Organs were obtained on day 7 post-infection. <b>A.</b> The numbers of donor cells recovered from recipient lungs and mediastinal lymph nodes (mdLN) on day 7 post-infection. <b>B.</b> Cells harvested from the lungs and mdLN were restimulated <i>in vitro</i> in the presence of SIINFEKL peptide and the production of IL-2 (top) and IFN-γ (bottom) by donor OT-I cells was quantified by flow cytometry. The percentage of positive events in stained samples and fluorescence minus one (FMO) negative controls are shown. Each data point represents an individual mouse and the bar represents the mean. Results from one of three experiments are shown. Statistical significance was tested using the Mann–Whitney test. <b>C.</b> FAP knockout and WT mice were intranasally infected with 50 pfu PR/8 virus and sera were harvested on day 21 post-infection. Neutralising anti-influenza antibody titres in the sera were measured using haemagglutination inhibition (HI) assay. Each data point represents an individual mouse and the bar represents the mean. Statistical significance was tested using Kruskal-Wallis test with Dunn’s multiple comparison test. * p<0.05, *** p<0.001, ns not significant.</p

An adoptive transfer model to study the regulation of monocytes during ECM.

<p>(<b>A</b>) CD8⁺ T cells or CD8⁻ splenocyte fraction (Sp) isolated by MACS from uninfected or PbA-infected C57BL/6 donor mice on day 7 p.i. were adoptively transferred into PbA-infected MacGreen×RAG^−/− recipient mice as depicted. Only primed CD8⁺ T cells (red circle) isolated from PbA-infected C57BL/6 donor mice induce NS (orange recipient). Representative data of 3 independent experiments is shown. (<b>B</b>) Dot plot shows purity of CD8⁺ T cells routinely obtained by MACS sorting. (<b>C</b>) MacGreen×RAG^−/− recipient mice receiving saline (n = 4 mice), naïve (n = 4 mice) or primed CD8⁺ T cells (n = 5 mice) or CD8⁻ splenocytes (n = 4 mice) were infected with PbA and clinical scores monitored daily, *p = 0.01 (unpaired <i>t</i> test). (<b>D</b>) Clinical score of each mouse on day 5–7 p.i. Each symbol represents one mouse. Recipients of naïve and primed CD8⁺ T cells are shown in green and red respectively. (<b>E</b>) Percent survival of MacGreen×RAG^−/− recipients after adoptive transfer, *p<0.05 (Mantel-Cox test).</p

Endothelium-interacting GFP⁺ leukocytes in the brain microvasculature are monocytes.

<p>PbA-infected MacGreen mice with NS were injected i.v. with 5 µg of either (<b>A</b>) an isotype control, (<b>B</b>) anti-Ly6C (n = 2) or (<b>C</b>) anti-Ly6G antibody (n = 2) 1 hour prior to mice undergoing intravital imaging of the brain microvasculature. Representative snapshots from each group are shown. Scale bars represent 30 µm in (A), 100 µm in (B) and 58 µm in (C). Blue represents SHG signals from extracellular matrix fibres. White arrows indicate direction of blood flow. To aid orientation, vascular walls are outlined. Cells stained with Ly6C antibody show an orange punctate stain that co-localises with GFP (red arrowheads = adherent cells; yellow arrowheads = rolling cells). A total of 37, 29 and 45 blood vessels were assessed for the isotype control, Ly6C and Ly6G groups respectively. Number of blood vessels per mouse: Isotype (n = 37), Ly6C (n = 14, 15) and Ly6G (n = 9, 36). Representative snapshots of intravital imaging of the dermis of Lysozyme-GFP mice after injecting 5 µg of (<b>D</b>) an isotype control, (<b>E</b>) anti-Ly6C or (<b>F</b>) anti-Ly6G antibody, as reported <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat.1004236-Ng1" target="_blank">[34]</a>. Number of blood vessels per mouse: Isotype (n = 12), Ly6C (n = 11) and Ly6G (n = 11). Cells stained with Ly6C show a punctate stain as in the brain. Cells stained with Ly6G have a red surface stain. Scale bars represent 60 µm in (D), 30 µm in (E) and 20 µm in (F). (<b>G</b>) Changes in blood monocyte populations during ECM. Blood monocytes were quantified by flow cytometry after collection from either uninfected (UI) or PbA-infected mice that had developed ES or NS (n = 4–5 mice/group). Symbols represent individual animals and bars represent means. *P<0.05, **p<0.01 (ANOVA with Dunnett's post-test vs. UI control). (<b>H</b>) Representative flow cytometric plots showing blood monocyte populations in UI, ES and NS mice. The percentage of Ly6C^lo and Ly6C^hi monocytes are indicated. Data are from a single experiment (n = 4–5 mice/group).</p

<i>Plasmodium</i>-primed CD8⁺ T cells induce monocyte accumulation in MacGreen×RAG^−/− mice.

<p>PbA-infected MacGreen×RAG^−/− mice that had received saline, CD8⁻ splenocytes, naïve or primed CD8⁺ T cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat-1004236-g004" target="_blank">Fig. 4A</a> underwent intravital imaging on day 7 p.i. (n = 3 mice/group). Representative snapshots show (<b>A</b>) nil, (<b>B</b>) moderate and (<b>C</b>) severe levels of monocyte accumulation in the blood vessels. Scale bars represent 60 µm. Migratory paths of monocytes are shown as purple tracks. Blood vessels are marked by infusion of TRITC-conjugated dextran. White arrows indicate direction of blood flow. (<b>D</b>) % blood vessels with nil, moderate and severe levels of monocyte accumulation in MacGreen×RAG^−/− and MacGreen mice. Average number of (<b>E</b>) adherent and (<b>F</b>) rolling leukocytes per mm² of endothelium. A total 46, 123, 82 and 104 blood vessels were assessed for recipients of saline, CD8⁻ splenocytes, naïve and primed CD8⁺ T cells respectively. Number of vessels assessed per mouse: saline (n = 10, 12, 24), CD8⁻ splenocytes (n = 45, 28, 25), naive CD8⁺ T cells (n = 60, 11, 11), primed CD8⁺ T cells (n = 49, 33, 22). Blood vessel numbers for MacGreen mice are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004236#ppat-1004236-g002" target="_blank">Fig. 2</a>. Data are a mean of 2–3 independent experiments. Bars represent mean ± SEM, ns, not significant, *p<0.05, **p<0.001, ***P = 0.0001 (ANOVA, Tukey's multiple comparisons test).</p

Visualizing the behaviour of GFP⁺ leukocytes in the brain microvasculature.

<p>PbA-infected MacGreen (n = 16) and MacGreen×RAG^−/− mice (n = 4) were monitored for (<b>A</b>) % survival to pre-defined clinical endpoint, (<b>B</b>) clinical score of each mouse on day 5–7 p.i., *p<0.05, ns, not significant (Mann-Whitney test), or (<b>C</b>) % parasitemia (n = 4 mice/group), ns, not significant, *p<0.05, **p<0.005 (One way ANOVA, Tukey's multiple comparisons test). Each symbol in (B) represents one mouse. Data are a mean of 3–6 independent experiments. MacGreen mice underwent intravital imaging of the brain microvasculature at various stages including when mice were uninfected (UI) (n = 4 mice), during ES on day 5–6 p.i. (n = 5 mice) and after they developed NS on day 7 p.i. (n = 3 mice). (<b>D</b>), (<b>E</b>), (<b>F</b>) Representative snapshots of 3–5 independent intravital imaging experiments shows nil, moderate and severe levels of leukocyte accumulation. Migratory paths of GFP⁺ leukocytes are shown as purple tracks. Blood vessels are marked by infusion of TRITC-conjugated dextran. Blue represents second harmonic generation (SHG) signals from extracellular matrix fibres. White arrows indicate direction of blood flow. Perivascular myeloid cells are indicated by red arrows. Scale bars represent 100 µm in (D), 60 µm in (E) and 28 µm in (F). (<b>G</b>) % blood vessels that have nil, moderate and severe levels of leukocyte accumulation in UI, ES and NS. (<b>H</b>) Average number of rolling and adherent leukocytes per mm² of endothelium. A total 89, 90 and 43 blood vessels were assessed for UI, ES and NS groups, respectively. Number of blood vessels assessed per mouse: UI (14, 30, 28, 17), ES (14, 15, 23, 22, 16) and NS (12, 25, 6). Bars represent mean ± SEM. ns, not significant, *p<0.05, **p<0.005, ****P<0.0001 (Kruskal-Wallis-Test, Dunn's multiple comparisons test).</p