macrophages

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Dissemination to other organ systems is also observed, and is potentially facilitated by macrophages. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the host and parasite during infection. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The parasite transcriptome reveals large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication and dissemination. Together, our data provide insights into the host response in primary human macrophages

Assays of Phagocytosis and Attachment.Phagocytosis and attachment of ligand-coated SE was measured as described (8).The results were scored with a phase-contrast microscope by counting the number of SE ingested or attached per 100 phagocytes.These numbers are termed phagocytic index and attachment index, respectively.In some experiments, ligand-coated glass beads were added with the ligand-coated SE.Under such conditions, attachment of SE to macrophages could not be scored by phase-contrast microscopy.In such cases, the following procedure was used.After a 45 min incubation of macrophages with both beads and SE, monolayers were washed, cooled to 0°C, and incubated for 30 min with fluoresceinated anti-SE.The monolayers were washed again, and attachment of SE was scored using fluorescence microscopy.The presence of beads that did not bear ligand (BIgM) caused a 15% depression in the attachment index of all ligand-coated SE.

Assay of Total [SH]20:4 Release.
Macrophages prelabelled with [SH]20:4 were washed four times in PD and were overlaid with 1 ml of serum-free MEM.Zymosan (160 ~g/ml) and BIgM, BIgMC, and BIgG (all at 5 mg/ml) were added as indicated.The cells were incubated at 37 °C in a 95% air, 5% COe atmosphere, and at the times indicated, aliquots of medium were removed and counted in Hydrofluor (National Diagnostics, Inc., Somerville NJ).The cells were washed in PD and scraped twice into 1 ml of Triton X-100.Portions of the cell iysates were assayed for radiolabel content, and protein was determined by the method of Lowry et al. (9) with bovine serum albumin as a standard.

"Activation" of Complement Receptors With Lymphokines.
To obtain cells that were capable of ingesting C3-coated particles, macrophage monolayers were incubated at 37°C forl6 h in supernatants containing a lymphokine secreted by appropriately triggered T lymphocytes (TCF) (generously provided by Dr. Frank Griffin, University of Alabama in Birmingham) (10).

Results
Macrophages were challenged with glass beads coated with a variety of ligands.Since IgM-coated glass surfaces do not promote the release of H~O~ from phagocytes (11), IgM-coated glass beads were chosen as a negative control.BIgM bound very well to macrophages, but they neither triggered release of 20:4 nor inhibited the release of 20:4 stimulated by zymosan (Fig. 1 A).In keeping with previous findings (4), IgG-coated beads did trigger the release of ~12% of cellular 20:4.Complement-coated beads caused no release of cellular 20:4 (Fig. 1A).
The inability of complement-coated beads (BIgMC) to stimulate release of 20:4 does not result from insufficient amounts of the ligand, C3.This was demonstrated by measuring the number of available C3 receptors on the cell surface following interaction with ligand-coated beads.While BIgM caused no reduction in either C3b or C3bi receptors, BIgMC caused a marked reduction (~85%) of both C3b and C3bi receptors (Table I).This reduction likely results from diffusion of receptors to the glass-adherent surface where they are occupied and trapped by interaction with the ligand.Thus, our observations suggest that the failure of macrophages to secrete 20:4 in response to BIgMC is not due to inefficient interaction between ligand and receptor, rather, ligated C3b and C3bi receptors are unable to stimulate secretion of 20:4.
The resident peritoneal macrophages employed in the above experiment have "inactive" complement receptors, i.e. receptors that do not promote phagocytosis of C3b-or C3bi-coated particles (12, 13, and   Monolayers of macrophages were cultured overnight in MEM plus 10% FCS, or TCF.The monolayers were then washed in PBS and incubated for 45 rain at 37 °C with a mixture of the indicated ligand-coated erythrocytes and ligand-coated beads as described in Materials and Methods.The monolayers were again washed in PBS and incubated at 0°C for 30 rain with fluoresceinated antibody directed against sheep erythrocytes.Attachment of erythrocytes to macrophages was scored using a fluorescent microscope.* Not done.Monolayers of macrophages were cultured overnight in MEM or TCF as described in Materials and Methods.The macropbages were washed and the phagocytic capacity was determined using the indicated ligand-coated erythrocytes.Results are expressed as phagocytic index, and the percent of phagocytes ingesting at least one erythrocyte is given in parentheses.Data are averaged from two separate experiments. be surprising that these receptors are incapable of promoting the release of 20:4.In order to measure the effect of active phagocytosis-promoting C3 receptors on the release of 20:4, we cultured the macrophages for 18 h in a source of an activating lymphokine (TCF).Griffin et al. (10,13) have shown that this lymphokine renders C3 receptors competent to promote phagocytosis, and Table II shows that complement-mediated phagocytosis is stimulated 7-fold by the conditions we employed.Nevertheless, BIgMC triggered no release of 20:4 from iymphokine-stimulated macrophages (Fig. 1 B).Thus, C3 receptors signal phagocytosis without signalling the release of 20:4 from peritoneal macrophages.Though C3 receptors of resident peritoneal macrophages may not themselves promote phagocytosis, Ehlenberger and Nussenzweig (12) have shown that C3 receptors can act synergistically with Fc receptors to promote phagocytosis.Therefore, we examined the ability of C3 receptors to augment the release of 20:4 stimulated by BIgG and zymosan.In unstimulated macrophages, saturating doses of BIgG and zymosan individually promote the release of 10-20% of cellular 20:4 (Fig. 1 A).A combined stimulus of zymosan plus BIgG provokes the release of an amount of 20:4 equal to the sum of the release triggered by either zymosan or BIgG alone (Fig. 1 A).This observation demonstrates that the engagement of either the Fc receptor, or those receptors that bind zymosan, does not exhaust the capacity of the macrophage to release 20:4, and that these receptors act in an additive manner.Engagement of C3 receptors by BIgMC, on the other hand, causes no augmentation of the release of 20:4 initiated by either BIgG or zymosan (Fig. 1 A).Thus, C3 receptors appear incapable of synergy with Fc receptors, or with those receptors that bind zymosan.
Even though treatment of macrophages with lymphokine does not enable the C3 receptors to promote the release of 20:4, it does induce the cells to secrete double the amount of 20:4 in response to zymosan (Fig. 1, A and B).In contrast, the Fc receptor-mediated release of 20:4 is unaffected by this treatment (Fig. 1,  A and B).

Discussion
Macrophages express receptors for the principle opsonic molecules in mammals, IgG and C3 (14).Both the Fc and C3 receptors are transmembrane glycoproteins which bind appropriately opsonized particles, and which are capable of initiating changes in cellular behavior (14).Ligation of Fc receptors by IgG-coated particles results not only in phagocytosis of the particle, but also in the concomitant release of H202 and 20:4 (2,14).C3 receptors differ from Fc receptors in several respects.While the Fc receptor is composed of a single polypeptide chain of ~55 kD (14), the C3 receptors are much larger.The C3b receptor is a 190 kD protein (15), and the C3bi receptor is composed of two polypeptide chains of 190 and 105 kD (16).Consonant with the structural difference between Fc and C3 receptors is their different behavior.C3 receptors can exist in two states, an inactive state, in which the C3 receptors bind C3coated particles but do not promote their ingestion, and an active state, in which particle binding is followed by phagocytosis (13).Resting macrophages bear inactive receptors that can be converted by the action of a iymphokine to the activated state (13).Second, while C3 receptors do promote phagocytosis, they TableII).Therefore, it may not Control cells receiving no stimulus (X) were similarly treated and incubated at 37°C in a 95% air, 5% COs atmosphere.At the specified times, duplicate aliquots of the medium were removed and counted.The cells were scraped twice in 0.05% Triton-X 100, and the radiolabel and protein content of the cell lysates were determined.Values are expressed as percent of total cellular 20:4 released into the medium, and are the mean of triplicate cultures.

TABLE I
Complement Receptors Bind to C3 on Complement-coated Glass Beads

TABLE II C3
Receptors of TCF-stimulated Macrophages Promote Phagocytosis