Cell Properties of Lung Tissue-Resident Macrophages Propagated by Co-Culture with Lung Fibroblastic Cells from C57BL/6 and BALB/c Mice

Abstract

Tissue-resident macrophages (Mø) originating from foetal precursors are maintained by self-renewal under tissue/organ-specific microenvironments (niches). We recently developed a simple propagation method applicable to tissue-resident Mø by co-culturing. Here, we examined the properties of lung tissue-resident Mø propagated by co-culturing with lung interstitial cells. The intracardially and intratracheally perfused lung from BALB/c and C57BL/6 mice could minimise the contamination of alveolar Mø and lung monocytes. Lung tissue-resident Mø could be largely propagated under standard culture media along with the propagation of lung interstitial cells demonstrating a fibroblastic morphology. Propagated lung Mø showed characteristic expression properties for Mø/monocyte markers: high expressions of CD11b, CD64 and CD206; substantial expressions of Mertk; and negative expressions of Ly6C, MHC II and Siglec-F. These properties fit with those of lung interstitial Mø of a certain population that can undergo self-renewal. Propagated fibroblastic cells by co-culturing with lung Mø possessed niche properties such as Csf1 and Tgfb1 expression. Propagated lung Mø from both the mouse types were polarised to an M2 phenotype highly expressing arginase 1 without M2 inducer treatment, whereas the M1 inducers significantly increased the iNOS-positive cell percentages in C57BL/6 mice relative to those in BALB/c mice. This is the first study to demonstrate fundamental properties of lung tissue-resident Mø propagated by co-culturing. Propagated lung Mø showing features of lung interstitial Mø can serve as an indispensable tool for investigating SARS-CoV-2 diseases, although lung interstitial Mø have gained little attention in terms of their involvement in SARS-CoV-2 disease pathology, in contrast to alveolar and recruited Mø.

Testicular Macrophages Produce Progesterone De Novo Promoted by cAMP and Inhibited by M1 Polarization Inducers

“…Previous studies have also revealed that (i) testicular interstitial cells and/or tissue-resident Mø produce CSF1, IGF1, and retinoic acid (RA) [ 10 , 12 , 26 ] along with AR and PGR [ 27 , 28 ], which are implicated in spermatogenesis, and (ii) certain tissue-resident Mø express GJA1 [ 29 , 30 ] along with TGFB1 and TGFBR2 [ 21 , 31 ]. Thus, we examined the expression of these molecules and RA synthetic enzymes, i.e., ALDHLA2 and RDH10, in Mø by RT-PCR.…”

“…We previously developed a propagation method for tissue-resident Mø for the liver, lung, spleen, and brain Mø of adult rodents [ 8 , 21 ]. By applying this method with modifications to the adult mouse testis, we successfully propagated testicular tissue-resident Mø by mixed culture with interstitial cells, largely composed of Leydig cells, in standard culture medium containing 10% FBS without additional growth factors.…”

“…Total RNA was isolated from the testes, testicular tissue-resident Mø, and interstitial cells in mixed culture using the TRI Reagent (TR118, Molecular Research Centre, Cincinnati, OH, USA); RT-PCR analysis was performed as previously described [ 21 , 22 ]. Total RNA (1 μg) was transcribed into first-strand cDNA using Moloney Murine Leukemia Virus (M-MLV) RT, RNase H − (316-08151, Nippon Gene, Toyama, Japan), and an oligo (dT) 18 primer, as per the manufacturer’s instructions.…”

Mixed-Culture Propagation of Uterine-Tissue-Resident Macrophages and Their Expression Properties of Steroidogenic Molecules

“…Recently, we developed a propagation method of tissue-resident Mø by mixed culture with the respective tissue/organ-residing cells that could be applicable for several tissue-resident Mø [ 21 ] and successfully propagated lung interstitial Mø [ 22 ], testicular interstitial Mø [ 23 ], and Kupffer cells [ 24 ], all of which showed identical properties in marker expressions to those in respective ex vivo Mø. In these mixed cultures, tissue-resident Mø could propagate along with the propagation of organ-specific interstitial cells, which behaved as the niche for the respective Mø because these interstitial cells expressed Csf1 , Csf2 , and/or Il34 as growth factors for Mø, as well as marker molecules specific for the respective niche cells.…”

Macrophages in Health and Non-Infectious Disease 2.0

“…Mice-originating tissue-resident macrophages specific for lungs were co-incubated with lung interstitial cells in order to study the microenvironment of the expression of specific markers for cell lines studied [ 11 ].…”