Cribriform Plate Microenvironment Assembles a Suppressive Myeloid Network during EAE-induced Neuroinflammation

Reviewer #1 (Public review):

Summary:

Laaker et al. investigate the immunological role of the cribriform plate during neuroinflammation using the EAE model. The authors combine immunohistochemistry, flow cytometry, and single-cell RNA sequencing to characterize CD11b+CD11c+ myeloid cells that accumulate at podoplanin (PDPN)-rich meningeal-lymphatic niches surrounding olfactory nerve bundles. They identified distinct populations of migratory dendritic cells (DCs) and macrophages retained at the cribriform plate that exhibit transcriptional signatures consistent with immune tolerance, reduced interferon signaling, and programmed cell death, including Pdcd1 (PD-1) expression. In parallel, CCR2+ monocytes and alternatively activated (M2-like) Arg1+/CHI3L3+ macrophages integrate into this niche, suggesting the establishment of a locally immunosuppressive myeloid network.

Strengths:

(1) Overall, the study postulates a novel model in which the cribriform plate functions as a specialized perineural immune interface that reshapes myeloid phenotypes during neuroinflammation.

(2) Suggests broader relevance for shaping peripheral immunity and therapeutic targeting. If DCs are being "tuned" at this exit site, it could influence what reaches cervical lymph nodes and how peripheral responses are set during CNS autoimmunity; the authors explicitly position this as relevant to CNS autoimmunity and possibly other CNS diseases (while acknowledging the need for human validation).

(3) Technical sound and highly original work. Convergent multi-method support: the central narrative is backed by immunohistochemistry + flow cytometry + scRNA-seq, rather than a single assay. The headline conclusion (tolerogenic/suppressive skew at the cribriform plate during EAE) is explicitly built from these combined modalities.

Weaknesses:

(1) In Figure 1, the manuscript would be strengthened by quantification of CSF1R-GFP+ and CD11c-eYFP+ cells in PDPN+LYVE1- versus PDPN+LYVE1+ regions in both control and EAE mice. This would demonstrate selective accumulation or retention of myeloid cells at the cribiform plate niche.

(2) While the PostContact-seq strategy is innovative (Figure 3), additional justification is needed to demonstrate that tissue dissociation did not artificially disrupt PDPN-myeloid contacts. The relatively small proportion of live PDPN-rich doublets (~2.5% total aggregates and ~18% PDPN+ within total aggregates) raises questions about representativeness compared with in situ observations. The authors should also more explicitly elaborate on why PostContact-seq was favored over alternative approaches such as PIC-seq.

(3) The authors stated that results regarding cell-cell interactions were integrated across four intercellular communication methodologies (Figure 4B), but this integration is not clearly described in either the Results or Method sections. This needs clarification. Moreover, the interaction analysis in Figure 4B seems to rely on TALKIEN, which does not incorporate prior ligand-receptor knowledge. Given the availability of widely used tools, such as CellChat and NicheNet, the authors may consider cross-referencing their findings.

(4) Given the increase in CCR2+ cells in PDPN+ regions (Figure S4), a pseudotime trajectory analysis may be valuable to test whether CCR2+ monocytes preferentially differentiate into CHI3L3+ immunosuppressive macrophages, PD-1+ DCs, or other myeloid subsets in post-contact versus no contact.

(5) Validation of immunosuppressive signatures in macrophages (Fig. 4G-H) using the same FACS-based post-contact versus no-contact sorting strategy (as in Figure 3A) would strengthen the conclusions.

(6) The identity of CD45IV+ cells in contact with PDPN+ cells is unclear (Figure 6B-C). The authors should provide a gating strategy demonstrating that these cells are CD11b+CD11c+ DCs within the PDPN+ doublet population, and ideally show whether these dying cells are PD-1+. Furthermore, co-labeling in tissue sections for PD-1, cleaved caspase-3, and CD11c-eYP would provide important spatial validation of flow cytometry findings (Figure 6E).

(7) In Figures 1F-H, the authors should comment on the morphological differences of CD11c+ cells in the olfactory bulb versus those infiltrating the cribriform plate.

Reviewer #2 (Public review):

Summary:

In this article, Laaker et al described diverse populations of macrophages and dendritic cells found in and around the cribriform plate in the context of a neuroinflammation caused by an autoimmune disease (EAE). The authors utilize elegant histochemical staining and a nifty approach to sort doublets to interrogate cells that are in contact with one another, presumably in vivo. Notably, they uncover a population of CD11c+CD11b+ cells interacting with M2 macrophages and PDPN+ fibroblasts and lymphatics. These cells are heterogenous but some of these DCs express PD-1, and transcriptional profiling suggests they may have immunosuppressive behavior. Altogether, this article explains well the complexity of cell populations found around the cribriform plate during inflammation, and is suggestive of different interactions that trigger these different phenotypes from immune cells.

Strengths:

Beautiful images of a unique CNS: peripheral interface that support a novel scRNA approach to understanding how different cell populations engage in functional interactions in vivo.

Weaknesses:

It's currently unclear how the sorted populations reflect in vivo interactions or a propensity to form aggregates during ex vivo processing. The authors address both podoplanin-expressing cells as stromal cells and as lymphatic endothelial cells, but at times it's unclear which of these two populations is being analyzed and which is the most relevant. While novel observations, most of these findings are descriptive and lack functional correlates, and in places, the potential implications could use further discussion.

Author response:

We would like to thank the reviewers for their supportive comments which largely agree with our main finding that a heterogeneous population of dendritic cells and Th2-skewed macrophages interact with the PDPN+ niche at the cribriform plate during EAE neuroinflammation. Additionally, they have provided several meaningful critiques to our study which we are now working on addressing in a newly revised manuscript.