Porcine Alveolar Macrophage-like cells are pro-inflammatory Pulmonary Intravascular Macrophages that produce large titers of Porcine Reproductive and Respiratory Syndrome Virus

Lung inflammation is frequently involved in respiratory conditions and it is strongly controlled by mononuclear phagocytes (MNP). We previously studied porcine lung MNP and described a new population of cells presenting all the features of alveolar macrophages (AM) except for their parenchymal location, that we named AM-like cells. Herein we showed that AM-like cells are macrophages phagocytosing blood-borne particles, in agreement with a pulmonary intravascular macrophages (PIM) identity. PIM have been described microscopically long time ago in species from the Laurasiatheria superorder such as bovine, swine, cats or cetaceans. We observed that PIM were more inflammatory than AM upon infection with the porcine reproductive and respiratory syndrome virus (PRRSV), a major swine pathogen. Moreover, whereas PRRSV was thought to mainly target AM, we observed that PIM were a major producer of virus. The PIM infection was more correlated with viremia in vivo than AM infection. Finally like AM, PIM-expressed genes were characteristic of an embryonic monocyte-derived macrophage population, whose turnover is independent of bone marrow-derived hematopoietic precursors. This last observation raised the interesting possibility that AM and PIM originate from the same lung precursor.


AM-like cells are Pulmonary Intravascular Macrophages (PIM). AM-like cells represented the sec-
ond-most abundant population of lung MNP after AM, being 10 times less represented than AM, but at least 5 times more numerous than cDC1, cDC2 or moDC 5 . In a first description of AM-like cells, we demonstrated that these cells were not located in the alveoli, and that our broncho-alveolar lavage procedure avoids the contamination of the parenchymal cell preparation by AM 4 . However, we were uncertain whether AM-like cells were localized in the lung interstitium or on the endothelial wall, in contact with the vascular lumen as described for PIM. Since PIM have been described as highly phagocytic cells 20 , an experiment was devised in order to label phagocytic cells having access to blood-borne particles using FITC-labelled pseudomonas bacteria injected in the jugular vein. Peripheral blood mononuclear cells (PBMC), broncho-alveolar lavage (BAL) and parenchyma were collected 10 minutes post-bacterial injection, cell suspensions were recovered and stained for cDC1, cDC2, moDC and AM/AM-like discrimination as previously described 4 (Fig. 1a). In the parenchyma, 19% +/− 4% of AM-like cells presented a clear phagocytosis of FITC-labelled bacteria, no cDC1 and few cDC2 were FITCstained. Interestingly, moDC presented a highly variable FITC staining, from 2% to 18% (mean 13% +/− 5%). In the BAL, no FITC staining either of AM (Fig. 1a,b) or of BAL DC (data not shown) was observed. In order to identify blood-circulating cells that might phagocytose bacteria and contaminate the parenchymal preparation, the same gating as for parenchymal cells was applied for PBMC. No events were observed that could correspond to AM-like cells (empty gate 4 in Fig. 1a), proving that AM-like cells were not blood circulating cells. Conversely, the PBMC gates 2 and 3 (Fig. 1a), corresponding respectively to cDC2 and moDC in the parenchymal gating, presented a strong phagocytosis of bacteria, since around 50% of these two cell types presented FITC staining. Thus, the FITC staining of parenchymal cDC2 and moDC might mostly be due to blood circulating cell contamination of the parenchyma. In order to demonstrate that the lower staining of AM, cDC1 and cDC2 was not due to an intrinsic defect in bacterial phagocytosis, BAL cells were incubated in vitro with FITC-stained pseudomonas. All the DC/Macrophages subtypes (cDC1, cDC2, moDC and AM) presented more than 45% bacteria phagocytosis (data not shown), in agreement with similar capacities of these cells to phagocyte bacteria, provided that they enter in contact with the pathogen.
Thus, AM-like cells are the only parenchymal residing MNP population consistently phagocytosing blood bacteria. Although only 20% of the AM-like cells appeared FITC positive, we consider that the 3 to 4 hours needed for MNP extraction from lung tissue allowed a majority of PIM that had phagocytosed bacteria to quench the acid-sensitive FITC, eventually exhibiting no staining. Alternatively, PIM might be able to move across the blood barrier and locate temporarily in the interstitium, losing their access to blood. We then proceeded to tissue staining and confocal imaging in order to visualize blood-borne bacterial phagocytosis in situ. We observed the localization of FITC-stained bacteria inside CD163 high /MHC-II high cells (AM-like cells) in the parenchyma, in ve-Cadherin (a blood vessel specific marker 21 ) rich area (Fig. 1c). Altogether, we show here that AM-like cells are bona fide PIM. express CCR2 and CX3CR1, unlike the other lung DC/Macrophages populations, whereas they both expressed high levels of MertK and CD64 4 . We then tested the differential expression of genes between macrophages derived from blood monocytes or from tissue resident embryonically-derived, self-renewable macrophages as described by Geissmann and Lambrecht teams 22,23 . Both AM and PIM/AM-like cells strongly expressed the markers differentially represented in embryonic-derived macrophages, HDAC10 and PU.1, whereas they expressed significantly lower amount of the hematopoietic cell marker c-Kit (Fig. 2a,b). This transcriptomic profile was highly distinctive from blood monocytes 24,25 and definitely excluded the possibility that PIM/AM-like cells were sequestered lung monocytes. Interestingly, MAFB, a gene involved in the decrease of macrophage proliferation during their differentiation 26 , was the only observed marker differentially expressed in PIM/AM-like cells and AM. The PIM/AM-like cells express higher levels than AM (Fig. 2). Like AM, porcine PIM/AM-like cells are likely embryonic-derived macrophages, whose renewal must be independent from blood monocytes. PIM/AM-like cells are infected, produce PRRSV and are pro-inflammatory upon in vitro infection. Because of the great similarities between AM and PIM/AM-like cells and since AM are thought to be the main target of PRRSV, we compared the susceptibility of AM and PIM/AM-like cells to PRRSV infection using the genotype 1.3 LENA virus 27 . AM and PIM/AM-like cells were cell-sorted and infected in vitro at low multiplicity of infection (MOI) 10 -3 . Then, 24 h later, intracellular viral RNA load and infectious viral particle titers were measured using RT-qPCR on the cell pellet and virus titration on cell culture supernatant. PIM/AM-like cells and AM show similar PRRSV RNA load (Fig. 3a) and PRRSV infectious particles production (Fig. 3b). However, AM presented a more variable susceptibility to in vitro PRRSV infection than PIM/AM-like cells. We repeated the same experiment using a high MOI in order to infect simultaneously a majority of macrophages; we measured the transcriptomic cytokine response of the cells, focusing on TFNα, IL-8 and IL-6 cytokines previously described as highly expressed in PIM [18][19][20] . PIM/AM-like cells significantly upregulated TNFα expression upon infection, whereas IL-8 and IL-6 were also upregulated, although non-significantly, highlighting the pro-inflammatory role of PIM/AM-like cells upon PRRSV infection (Fig. 3c).  28 . Moreover, in agreement with this result, the proportion of AM and PIM/AM-like cells among MHC-II high BAL and parenchymal cells respectively decrease (Fig. 4b). The amount of PRRS viral RNA was then measured by RT-qPCR on sorted cells (Fig. 4c). Both AM and PIM/AM-like cells were positive for viral RNA. A correlation analysis was performed with the following parameters: AM and PIM/AM-like cells PRRSV infection titers (Fig. 4c), serum PRRSV titers (viremia, as depicted in 29 ) as well as whole lung tissue viral load (Fig. 4d). PIM, but not AM infection, appeared correlated with lung titer (0.99 and 0.0025 respectively), and with serum titer (0.76 and −0.53 respectively) (Fig. 4e). This correlation must be taken with caution because it mainly stands on one individual presenting high viral titer in blood, lung and PIM/AM-like cells but not in AM.

In vivo, PRRSV infection of PIM/AM-like cells correlates with lung and blood PRRSV titer. Our
We finally measured by RT-qPCR, the in vivo cytokine response of AM and PIM/AM-like cells in mock and PRRSV-infected animals at 10 dpi. No cytokine presented a significant differential expression when conditions or cell types were compared (Fig. 5a). In order to assess the global AM/PIM responses, we then ran a principal component analysis (PCA) on the cytokine data, comparing AM and PIM/AM-like cells in non-infected or infected conditions. PIM/AM-like cells produced more cytokine mRNA than AM except for IL-4 in mock infected animals, and for IFNβ, IL-12p35, IL-12p40/IL-23 and IL-4 in infected animals. In infected animals, the first axis of the PCA encompassed 39% of the total variability of the samples (Fig. 5b). It segregated IL-8 and TNFα from IFNα, IL-6, TGFβ, IL-4, IL-12p35 and IL-12p40/IL23 cytokines. Except for the inter-individual variations, no obvious interpretation for this splitting can be proposed. Interestingly the second axis still encompassed 30% of the variability and clearly segregated PIM/AM-like cells from AM. Indeed PIM/AM-like cells expressed more IL-8, TNFα, IL-6 and IFNα, whereas AM expressed more IL-12p35 and IL-4. Thus we could observe in vivo, a tendency of PIM to produce more cytokines than AM at steady state and upon infection. However globally, at 10dpi, both PIM/AM-like cells and AM responded weakly to PRRSV.

Discussion
Herein, we showed that the previously described porcine AM-like cells 4 phagocytosed blood-borne bacteria and produced inflammatory cytokines such as TNFα, IL-8 and IL-6 18 . Some teams succeeded in isolating PIM using perfusion of the pulmonary vasculature with collagenase solution 30 . Using this technique, the best controlled experiment 31 measured a PIM/AM ratio of 1:10, in perfect agreement with the AM-like/AM ratio we previously observed 5 . Despite our inability to stain all AM-like cells, this last data associated with the homogeneity of AM-like cell population phenotype 4 are in strong support with the whole AM-like cell population being bona fide PIM.
Thanawongnuwech observed 20 years ago that collagenase-isolated PIM could be infected by PRRSV 31 , moreover in vivo PRRSV infection decreased the ability of the lung to retain copper particles 32  in the blood vessel lumen, allowing them to shed virus directly in the blood circulation, they might likely be the major cell types responsible for viremia.
Interestingly, we also showed here that both porcine AM and PIM/AM-like cells harbored characteristics suggesting that they derive from embryonic monocytes rather than from adult bone marrow, in consistency with what has been demonstrated for mouse AM 22,23 . Although surprising according to their location in contact with blood, PIM origin can be paralleled with the recently described resident arterial macrophages in mice 33 . These cells are embryonic monocyte-derived, and self-renew in the aortic wall 33 . It has been shown that PIM had an important role in xenogeneic graft failure, and that their depletion before pig lung graft to primate extended the duration of graft functionality 34 . The embryonic origin of PIM implies that the lung vasculature wall of the PIM have been described in the species from the Laurasiatheria superorder whereas no PIM were observed in primates and mice, species belonging to the Euarchontoglires superorder 6,7,9,14 . However, in rats and humans, evidence exist of a few constitutive PIM that increase in number upon liver dysfunctions [35][36][37] . In the rat model, PIM induced upon bile-duct ligation were shown to be responsible for endotoxin-induced mortality 38 . It would thus be interesting to re-examine the role of PIM in the liver-induced inflammatory medical conditions. Interestingly, it has been shown in lambs that PIM and AM appeared in the developing lung within the same time frame, increasing progressively from 1 day to 3 weeks of age 39 . It is tempting to speculate that AM and PIM originate from the same lung cell precursor that relocate after birth either in the alveoli or the lung endothelium. This precursor would have, even in Euarchontoglires, the potential to differentiate in PIM, provided the appropriate, still unknown, endothelial/blood stimulus is present.
It is worth noting that AM express lower MAFB mRNA than PIM/AM-like cells. MAFB has been described as a repressor of cell-renewal capacities in adult macrophages 40 . Thus the higher MAFB expression in PIM/AM-like cells would be in agreement with a lower proliferation capacity compare to AM, raising the interesting possibility that PIM/AM-like cells would originate from AM.

Materials and Methods
Pseudomonas FITC-staining. 1.10 10 cfu of UV-inactivated pseudomonas were resuspended at 2 mg/ml in 0.1 M Na 2 CO 3 pH9 buffer and 150 µg of fluorescein isothiocyanate (FITC) (SIGMA) per ml of bacteria were added and incubated for 2h at 4 °C. Bacteria (FITC-pseudomonas) were then washed twice in PBS and frozen in glycerol before use.

Pig lung cells collection.
For in vitro characterization and infection, lung tissue samples were obtained from 5-to 7-month-old Large White conventionally bred sows from UEPAO, Tours, France. A BAL procedure was performed twice on the isolated left lung with 250 ml of PBS supplemented with 2mM EDTA (PBS/EDTA), to collect AM. Next, a 1-cm slice of external lung parenchyma was dissected from the same lung. Tissues were minced and incubated in nonculture-treated Petri dishes, to avoid differential plastic adherence of MΦ and DC, for 2 h at 37 °C in complete RPMI, consisting of RPMI 1640 supplemented with 100 IU/ml penicillin, 100 mg/ml were catheterized under gaseous anaesthesia in order to reach the right atrium. Before FITC-pseudomonas injection, 30 ml of blood was withdrawn in heparinized tube as negative control. 0.6 to 10 mg of FITC-pseudomonas were resuspended in 5 ml physiological serum and injected in the catheter. After 10 minutes, 30 ml of blood was withdraw in heparinized tube as positive control. The animal was euthanized and the lung was sampled. An extensive BAL was processed using 3 times 100 ml PBS/EDTA. Parenchyma was collected, a 1 cm piece was frozen in Tissue Teck (Sakura, Paris, France) and conserved at −80 °C, and the remaining tissue was minced and digested in Collagenase/Dispase/DNase medium, and single cell suspension was retrieved as previously described 4 . BAL and parenchymal cell suspensions were then frozen in FCS/10% DMSO. Blood cells were ficolled and PBMC were then frozen in FCS/10% DMSO.
Flow cytometry analysis and cell sorting. Pig cell surface staining was performed as previously described 4 , using anti-MHC-II (clone MSA3) and anti-CD172a (clone 74-22-15a) (monoclonal antibody center Washington State University -Pullmann, WA), unlabelled or Phycoerythrin (PE) coupled anti-CD163 (clone 2A10/11) from Serotec (Oxford, UK) and isotype matched secondary antibodies coupled to PE, Alexa-488, and Alexa-647 (Invitrogen). Infected cells were then fixed in 4% paraformaldehyde before flow cytometry analysis. Samples were acquired on a Fortessa (BD-Bioscience) or sorted on a MoFlo ASTRIOS (Beckman-Coulter). For sorting, preparations were enriched in DC/Macrophages by gradient (Optiprep; Nycomed Pharma). Acquired data were analysed using FlowJo software (version X.0.6). Cryosections were fixed in methanol/acetone (1:1) at −20 °C for 20 min. Fixed slides were saturated using PBS supplemented with 5% horse serum (HS) and 5% swine serum (SS) 30 min at room temperature (RT). The same primary antibodies anti-MHC-II, anti-CD163 as in flow cytometry were used as well as anti-FITC (Fluorescein/ Oregon Green Polyclonal Antibody, Alexa Fluor 488, ThermoFisher scientific) and a poly-clonal rabbit anti-ve-Cadherin (H-72, Santa-Cruz Biotechnology, UK), and secondary antibodies were added at 4 °C overnight or 30 min, respectively. Slides were analyzed using an LSM510/U700 confocal microscope (Zeiss, LePecq, France). Cell infection and viral titration. Sorted populations were cultured in complete RPMI for 24 h in flat-bottom 96-well plates with 3.10 5 cells/well and then infected at a MOI of 10 −3 with a PRRSV virus (LENA) in complete RPMI. At 24hpi, supernatants were collected and frozen at −80 °C. After washing, cells were lysed in 100µL of RNA extraction buffer from the Arcturus PicoPure RNA Isolation kit (Life Technologies) for quantification of viral RNA by RT-qPCR. The same protocol was applied with a MOI of 5 for quantification of cytokine production by RT-qPCR. PRRSV titers (TCID50/ml) were assessed according to the OIE manual of diagnostic test. RNA extraction. Total RNA from sorted cells or infected cells were extracted using the Arcturus PicoPure RNA Isolation kit according to the manufacturer's instructions. Contaminating genomic DNA was removed using a Qiagen RNase free DNase set.
Real-time quantitative PCR (qPCR). RNA was reverse transcribed using random hexamers and the Multiscribe reverse transcriptase (Life Technologies). qPCR were performed as previously described 4  For each gene, data were normalized to the reference gene RPS24 (Ribosomal Protein S24) expression and presented as relative expression (arbitrary units (AU)). In Fig. 2, for each animal, the population with the highest expression was considered as 100 and the other populations were normalized to it. Each symbol represents one animal.

In vivo infection and tissue collection. For in vivo PRRSV infections, experiments were performed
at ANSES (Ploufragan, France). The animal experiment was authorized by the French Ministry for Research (authorization no. 2015060113297443) and approved by the national ethics committee (authorization no. 07/07/15). Eight specific pathogen-free (SPF) pigs (free from PRRSV, Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae) were housed in biosecurity level-3 air-filtered animal facilities. Treatments, housing, and husbandry conditions conformed to the European Union Guidelines (Directive 2010/63/EU on the protection of animals used for scientific purposes). At 10 weeks of age, 4 pigs were inoculated intranasally with LENA strain (5.10 5 TCID50/per animal in 2.5 ml per nostril). Ten days post infection, animals were anesthetized (Zoletil, Virbac, France) and exsanguinated. BAL cells were collected using 500 ml PBS/EDTA for right and left diaphragmatic lobes. BAL and parenchymal cells were sampled as above (pig lung cells collection). Cells were washed in PBS/EDTA and frozen in FBS/10% DMSO. A part of the data issued from this in vivo infection has already been published in 28 . Statistical analysis. All data were analysed using Graph Pad Prism (version 6) for Mann-Whitney test; R software (version 3.4.0) and package FactoMineR (version 1.39) for Principal Component Analysis method (PCA) and Pearson correlation coefficient determinations. When scatter plots are used, the mean is depicted by a horizontal bar. Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.