In the Hamerman Lab, we are interested in understanding how myeloid cells contribute to both productive and pathological immune responses during infection, inflammatory, and autoimmune diseases.

Our research focuses on monocytes and macrophages, and conventional and plasmacytoid dendritic cells, key players in innate immune responses that set the stage for subsequent adaptive immunity.

We are particularly interested in understanding how signaling by Toll-like receptors (TLRs) is regulated in these innate cells and how dysregulated TLR responses contribute to both initiation and propagation of inflammatory and autoimmune diseases, including systemic lupus erythematosus (SLE) and the autoimmune complication macrophage activation syndrome (MAS).

We also have a key interest in monocyte and macrophage development during homeostasis, and how this process changes during inflammation, whether due to infection, inflammatory or autoimmune diseases.

Our research will lead to a better mechanistic understanding of how TLRs and myeloid cells function and will allow for identification of new therapeutic intervention points in inflammatory and autoimmune diseases.

Flightless-1 in lung macrophage and DC development and function

In myeloid cells, the actin cytoskeleton controls important functions including adhesion, migration, and phagocytosis. Actin capping proteins both positively and negatively regulate the actin cytoskeleton, with both unique and overlapping functions.

We have a particular interest in Flightless-1, a member of the gelsolin family of actin capping proteins that has principally been studied in non-hematopoietic cells. In macrophages cells, Flightless-1 is a negative regulator of the NLRP3 and NLRC4 inflammasomes, which we showed depends upon the signaling adaptor BCAP. To investigate the role of Flightless-1 in myeloid cells in vivo, we generated a conditional knockout allele and deleted in either monocytes, macrophages, and neutrophils or in dendritic cells (DCs), with a goal to investigate how Flightless-1 regulates key actin-dependent processes in these cells. For these studies, we focused on macrophages and conventional DCs (cDCs) in the lung, which are situated to respond to respiratory pathogens.

In the lung, alveolar macrophages (AMs) are uniquely situated in the alveoli and are the only tissue macrophage found outside the body’s epithelial barrier. AMs have both homeostatic and immune functions—to keep alveoli clear of debris to facilitate gas exchange and to phagocytose pathogens and initiate inflammation. Underscoring the importance of AMs, deficiency in these cells causes pulmonary alveolar proteinosis (PAP), a disease where the alveoli fill with surfactant and cellular debris causing impaired breathing, and, in severe cases, progressive respiratory failure.

AMs differentiate from fetal monocytes during a short perinatal window supported by the cytokine GM-CSF. During this process monocytes become pre-AMs in the lung parenchyma and then cross the alveolar epithelium to finish their maturation in the alveoli. AM differentiation requires cell adhesion and migration for AM progenitors to become juxtaposed to the alveolar epithelium to receive the appropriate GM-CSF signals for differentiation and to perform the difficult task of crossing the epithelial barrier. We find that deletion of Flightless-1 in monocytes causes a block in AM differentiation with pre-AMs accumulating in the lung parenchyma and unable to cross into the alveoli. GM-CSF upregulates Flightless-1 expression in Ly6Chi monocytes, and Flightless-1 deficiency in these cells causes changes in cell size and shape and the actin cytoskeleton. Therefore, we have uncovered a new role for this actin capping protein in alveolar macrophage differentiation and identified an important function of GM-CSF in regulating the actin cytoskeleton in monocytes through Flightless-1. We are currently investigating precisely how Flightless-1 regulates monocyte and pre-AM adhesion and migration, as well as how it regulates mature AM function and other tissue macrophage populations.

cDCs in the lung are critical for detecting and internalizing pathogens in the airways and then migrating to the draining lymph nodes where they present antigens to T cells. The actin cytoskeleton can regulate all of these key processes. We found that phagocytosis of bacteria and fungal particles in vitro was defective in the absence of Flightless-1. In vivo, deletion of Flightless-1 in cDCs resulted in reduced phagocytosis by cDC1s in the lung and reduced migration to the draining mediastinal lymph node. We additionally found improper positioning of cDC1s further away from airways in lungs from mice lacking Flightless-1 compared to controls in the steady state. Thus, Flightless-1 regulation of actin dynamics is critical in regulating lung cDC1 positioning, phagocytosis, and migration. We are continuing to investigate how Flightless-1 controls cDC function in the lung and other tissues.

Monocyte-derived inflammatory hemophagocytes in disease

Monocytes are innate immune cells that develop in the bone marrow and are continually released into circulation, where they are poised to enter tissues in response to homeostatic or inflammatory cues. Monocytes are highly plastic cells that can differentiate in tissues into a variety of monocyte-derived cells to replace resident tissue macrophages, promote inflammatory responses, or resolution of inflammation.

We identified a unique monocyte differentiation pathway for cells specialized for the phagocytosis of red blood cells during sustained systemic inflammation. We first identified these inflammatory hemophagocytes (iHPCs) in a mouse model of the autoimmune disease systemic lupus erythematosus (SLE) driven by transgenic overexpression of the innate endosomal RNA sensor TLR7.1. These TLR7.1 mice develop severe anemia and thrombocytopenia, reminiscent of a complication of some autoimmune diseases called Macrophage Activation Syndrome (MAS). We found iHPCs differentiated from Ly6Chi monocytes and were found in multiple blood-rich organs, including the blood, spleen, liver, and bone marrow. In TLR7.1 mice iHPC numbers correlated with anemia and thrombocytopenia in this lupus-associated MAS model, and depletion of Ly6Chi monocytes led to a rescue from MAS.

We investigated additional highly inflammatory diseases associated with anemia and we also found iHPCs differentiate in a model of severe malarial anemia caused by blood stage infection with Plasmodium yoelii where they require MyD88 and endosomal TLRs for differentiation. Therefore, during several inflammatory anemias iHPCs differentiate from monocytes and contribute to pathology.

Current projects in the lab aim to understand iHPC differentiation and function in both MAS and during severe malarial anemia. In mouse lupus-associated MAS, we are investigating signals downstream of TLR7 that promote iHPC differentiation, including the transcription factor IRF5—strongly associated with risk of lupus and other autoimmune diseases as well as important for differentiation of a variety of inflammatory macrophages.

We are also investigating human MAS associated with systemic juvenile idiopathic arthritis, the autoimmune disease where this syndrome is most frequently seen. In children with MAS, we are using single cell approaches to study circulating monocyte phenotypes, including hemophagocytes to better understand monocyte contributions to this serious disease.

Lastly, we want to understand iHPCs in severe malarial anemia using the Plasmodium yoelii 17XNL model, including their differentiation pathways, phagocytic specificities for red blood cells, and parasites, and participation in anemia. We hope our studies will lead to a better mechanistic understanding of how monocytes contribute to these diverse pathologies and help identify therapeutic targets to restrain disease.

Immune complex activation of pDC IFNα production in lupus

Plasmacytoid dendritic cells (pDCs) are innate immune cells specialized for responding to internalized nucleic acids via endosomally-localized TLR7 and TLR9, resulting in potent IFNα production. During viral infection, IFNα production by pDCs is protective. However, pDC production of IFNα is pathogenic in the autoimmune disease Systemic Lupus Erythematosus (SLE), where IFNα and signatures of this cytokine family are elevated and correlate with disease activity and severity. SLE is also characterized by a loss of tolerance to nuclear antigens, resulting in the production of circulating autoantibodies to DNA, RNA, and nucleic acid binding proteins. These autoantibodies form immune complexes with nuclear antigens released from dying cells, which can be internalized by immune cells expressing receptors for the Fc portions of the autoantibodies in the complexes (FcRs). pDCs internalization of immune complexes in SLE leads to potent IFNα secretion, and this IFNα amplifies disease through its pleiotropic effects on the immune response, including increasing plasmablast differentiation and antibody production, leading to a feed-forward loop between B cells, antibodies, and pDCs that exacerbates disease.

Most studies of autoantibodies in SLE focus on IgG antibodies as this is the major circulating antibody isotype. Additionally, pDC IFNα secretion in response to SLE immune complexes depends upon recognition of IgG antibodies by FcgRIIa (CD32a). However, anti-nuclear antibodies (ANAs) of other isotypes, such as IgA and IgE have been documented but not well studied. Our work has focused on IgA autoantibodies in SLE. We have found a previously unrecognized role of IgA and its receptor FcαR (CD89) on pDCs in recognizing immune complexes made with autoantibodies from individuals with SLE when complexed with RNA-containing Smith ribonucleoproteins.

Our work shows a potent synergy between IgG and IgA autoantibodies allowing for more efficient immune complex binding and internalization and subsequent IFNα secretion by pDCs. In conjunction with these findings, we also found that pDCs from SLE donors had greater immune complex internalization than those from control donors, that this internalization correlated with surface FcαR expression, and that pDCs from SLE donors expressed more FcαR than from control donors.

Together, these findings suggest that immune complexes containing both IgA and IgG are important in driving SLE disease. We are currently investigating the mechanisms for synergy between FcαR and FcgRIIa, in pDCs, the regulation of FcαR expression on pDCs in SLE, and defining the spectrum of IgA autoantibody specificities in SLE and how they are associated with disease manifestations.