TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity
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eLife assessment
This important work advances our understanding of TLR7 signaling at epithelial surfaces that drives monocytes expansion and its impact on viral responses. The evidence supporting this conclusion is solid, particularly data demonstrating TLR7 stimulation and the requirement for TLR7 in the monocyte expansion. The work will be of interest to immunologists and virologists.
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Abstract
Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.
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eLife assessment
This important work advances our understanding of TLR7 signaling at epithelial surfaces that drives monocytes expansion and its impact on viral responses. The evidence supporting this conclusion is solid, particularly data demonstrating TLR7 stimulation and the requirement for TLR7 in the monocyte expansion. The work will be of interest to immunologists and virologists.
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Reviewer #1 (Public Review):
Jackson and Giacomassi et al. investigated the impact of repeated topical application of the TLR-7/8 agonist R848, mimicking single-stranded RNA viral infection, on circulating monocytes. Interestingly in this murine model of skin inflammation, they find that there is a striking increase in vascular patrolling (Ly6Clow) monocytes in the blood. In the majority of inflammatory settings so far described, it is the classical (Ly6Chi) monocyte population that is augmented. They found that this Ly6Clow monocyte expansion occurred in response to stimulation by R848 at epithelial barrier surfaces (skin and gut) and not following systemic administration of R848. Of note, the Ly6Clow increase was not dependent on type I or type II IFNs or CCR2, all factors that are important for Ly6Chi monocyte expansion in response …
Reviewer #1 (Public Review):
Jackson and Giacomassi et al. investigated the impact of repeated topical application of the TLR-7/8 agonist R848, mimicking single-stranded RNA viral infection, on circulating monocytes. Interestingly in this murine model of skin inflammation, they find that there is a striking increase in vascular patrolling (Ly6Clow) monocytes in the blood. In the majority of inflammatory settings so far described, it is the classical (Ly6Chi) monocyte population that is augmented. They found that this Ly6Clow monocyte expansion occurred in response to stimulation by R848 at epithelial barrier surfaces (skin and gut) and not following systemic administration of R848. Of note, the Ly6Clow increase was not dependent on type I or type II IFNs or CCR2, all factors that are important for Ly6Chi monocyte expansion in response to life-threatening infections, such as Toxoplasma gondii. Positive factors driving Ly6Clow augmentation are not identified. Alterations to circulating monocytes may have implications for secondary infection as R848-treated animals were less susceptible to flu infection. This research furthers our understanding of how tissues and organs have distinct mechanisms of communication in response to inflammatory and infectious stimuli and the implications this can have on circulating immune populations.
The conclusions of this paper are generally well supported by the data presented, however, some aspects of the study need to be clarified or extended. Additionally, some of the findings could be better discussed in the context of the current literature.
CSF-1 availability is described, initially by Yona et al. (DOI: 10.1016/j.immuni.2012.12.001), as an important factor extending the half-life of Ly6Clow monocytes in circulation. Given the expansion of Ly6Clow monocytes and their upregulation of CD115 in circulation, it would have been relevant to measure CSF-1 to assess whether this may be a candidate factor for the phenotype observed.
The conclusion that the altered monocyte compartment enhances protection against secondary infection is underdeveloped. The key experiment presented involves treating animals with R848 and demonstrating that they have an altered response to flu infection. This approach does not specifically assess the importance of monocytes. From these studies, it is only possible to conclude there is an association between monocyte alterations and secondary infection.
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Reviewer #2 (Public Review):
Jackson et al present a study focused on the role of TLR7 in emergency myelopoiesis following infection or injury. The investigators observe that TLR7 stimulation to the skin with the TLR7 agonist R848 causes an increase in circulating monocytes. This effect appears to require stimulation at an epithelial surface as it occurred with skin or intestinal administration but not intraperitoneal or intravenous administration. They demonstrate TLR7 specificity using TLR7-/- mice and the requirement for TLR7 expression in hematopoietic cells, likely myeloid cells. To determine if other TLR ligands can stimulate myelopoiesis, they compared skin administration with other TLR ligands (LPS, Poly I:C, CpG) or a general pro-inflammatory stimuli (TPA). None of these resulted in increased myelopoiesis, further highlighting …
Reviewer #2 (Public Review):
Jackson et al present a study focused on the role of TLR7 in emergency myelopoiesis following infection or injury. The investigators observe that TLR7 stimulation to the skin with the TLR7 agonist R848 causes an increase in circulating monocytes. This effect appears to require stimulation at an epithelial surface as it occurred with skin or intestinal administration but not intraperitoneal or intravenous administration. They demonstrate TLR7 specificity using TLR7-/- mice and the requirement for TLR7 expression in hematopoietic cells, likely myeloid cells. To determine if other TLR ligands can stimulate myelopoiesis, they compared skin administration with other TLR ligands (LPS, Poly I:C, CpG) or a general pro-inflammatory stimuli (TPA). None of these resulted in increased myelopoiesis, further highlighting TLR7 specificity. They confirm that this TLR7-mediated myelopoiesis occurs in the bone marrow as opposed to extramedullary sites (i.e. spleen) and differentiation occurs through the HSPC-MDP-cMoP pathway as opposed to a GMP-mediated differentiation. In addition to myelopoiesis, they demonstrate that R848 facilitates the transition of Ly6c high monocytes to Ly6c-low monocytes and tissue macrophages and this effect requires the Ly6c high monocytes. Furthermore, these effects occur independently of Ccr2 and Cx3cr1, known monocyte chemoattractant receptors. Finally, they identified that R848 administration enhances anti-viral responses. In mice topically treated with R848, they then exposed these mice to RSV and/or influenza. They observed that the R848 treated mice had reduced viral responses (defined by a decrease in weight loss and reduced viral replication). Overall, the data support that TLR7 administration to epithelial surfaces drives an increase in circulating monocytes, and this required TLR7 expression in myeloid cells. This is an interesting study that has implications for our understanding of how immune signals at peripheral sites drive the expansion of monocytes required to respond to infections and/or inflammation.
The conclusions are largely supported by the data, and several aspects of TLR7-mediated myelopoiesis are explored. However, there are some limitations to the data that need to be considered and reduce the generalizability of the conclusions made by the authors.
1. Data convincingly demonstrates that skin administration of the TLR7 agonist R848 causes an increase in circulating monocytes, particularly Ly6c low monocytes. In addition, this requires TLR7 expression and specifically TLR7 expression on myeloid cells. However, this raises an important question that is not answered by the present investigations. Specifically, the connection between local TLR7 administration requiring myeloid cells and how this directly leads to emergency myelopoiesis. Presumably, there is some factor released from local myeloid cells that then stimulates the bone marrow response and then a response that leads to the differentiation of Ly6c high monocytes to Ly6c low monocytes and infiltrating tissue macrophages. It is not clear if this is one factor or several factors. Presumably, this would be a circulating factor, though this is also not clear from the data. This appears to be a critical piece to tie in the connection between local TLR7 and emergency myelopoiesis. Furthermore, it is not clear how the dermal administration of R848 impacts the skin and if this is a critical feature of the response. Presumably, it generates local inflammation as evidenced by the data in 3C showing the proportions of monocytes and neutrophils. However, the impact on skin structure/function is not clear nor is there a definition of how this changes over the time course of the treatments.
2. The requirement for TLR7 stimulation on the skin is convincing. However, it is not clear how generalizable it is to all epithelial surfaces. The authors administer R848 in the drinking water and this causes myelopoiesis. However, the data supporting this as a direct effect of intestinal epithelial exposure is not explicitly demonstrated. The data using IP injections would seem to suggest that this is not a generic "epithelial surface response". IP injections are an administration to the peritoneum, an epithelial surface. The lack of an IP injection response would seem to argue that TLR7 responses are only to specific epithelial surfaces. This limits the generalizability of the observation. Alternatively, differences could be attributed to differences in TLR7 doses required at the distinct epithelial barrier sites. Further exploration of the specific epithelial interface requirements would provide better insight into the specific mechanism of how TLR7 stimulation works.
3. The authors demonstrate that dermal TLR7 and not other TLR ligands cause the increase in monocytes. Though the data is convincing for TLR7, the lack of a response with the other TLR ligands requires additional experiments to clarify if this is really TLR7-specific. Specifically, dose ranging experiments are needed to clarify if a lack of effect is simply due to differences in the sensitivity of TLR ligands to dermal exposure as opposed to being a TLR7 only effect.
4. The evidence of increased Ly6C low monocytes following dermal TLR7 in CCR2 null mice is intriguing. This suggests that TLR7-mediated emergency myelopoiesis is occurring independently of CCR2. However, this data is confusing as the authors also report that Ly6C low monocytes are generated from a Ly6C high monocyte intermediate. The data in Figure 6A supports that CCR2 null mice have baseline monocytopenia (a known feature of these mice) and then fail to generate Ly6C high monocytes following R848 exposure. Then how does this lead to an increase in Ly6C low monocytes if there are no Ly6C high monocytes as shown in the third panel of 6A? This is not clarified but critical to making this argument. There are also missing vehicle controls that would be important to interpreting these provocative results.
5. Data is lacking for direct TLR7 effects on the lung. These would appear to be important, given the focus on RSV and influenza responses in the study. As presented, the TLR7 protection from respiratory viral responses is via dermal TLR7 exposure followed by respiratory viral infection. This is unlikely to be clinically relevant, raising the significance of this model to human respiratory viral infection. An improved experimental design would incorporate respiratory TLR7 stimulation followed by pathogen exposure. In addition, given the focus on monocytes and macrophages, elucidating the impact on monocytes and lung macrophages, prior to and following infection would better define the connection between TLR7 exposure at epithelial barrier sites, emergency myelopoiesis, and respiratory viral infection.
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