Research ArticleImmunology Open Access | 10.1172/JCI179527

Xianpeng Liu,1 Ziyou Ren,2 Can Tan,3 Félix L. Núñez-Santana,1 Megan E. Kelly,1 Yuanqing Yan,1 Haiying Sun,1 Hiam Abdala-Valencia,4 Wenbin Yang,1 Qiang Wu,1 Takahide Toyoda,1 Marija Milisav,1 S. Marina Casalino-Matsuda,4 Emilia Lecuona,1 Emily Jeong Cerier,1 Lena J. Heung,5 Mohamed E. Abazeed,6 Harris Perlman,7 Ruli Gao,8 Navdeep S. Chandel,4 G.R. Scott Budinger,4 and Ankit Bharat1,4

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Liu, X. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Ren, Z. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Tan, C. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Núñez-Santana, F. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Kelly, M. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Yan, Y. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Sun, H. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Abdala-Valencia, H. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Yang, W. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Wu, Q. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Toyoda, T. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Milisav, M. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Casalino-Matsuda, S. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Lecuona, E. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Cerier, E. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Heung, L. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Abazeed, M. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Perlman, H. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Gao, R. in: JCI | PubMed | Google Scholar

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Chandel, N. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Budinger, G. in: JCI | PubMed | Google Scholar |

1Division of Thoracic Surgery/Canning Thoracic Institute, Feinberg School of Medicine, Northwestern University/Northwestern Medicine, Chicago, Illinois, USA.

2Department of Dermatology,

3Division of Cardiology, Department of Medicine, and

4Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

5Division of Infectious Diseases, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.

6Department of Radiation Oncology,

7Division of Rheumatology, and

8Department of Biochemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Address correspondence to: Ankit Bharat, 676 St. Clair Ave. Ste 650, Chicago, Illinois 60611, USA. Phone: 312.926.7552; Email: ankit.bharat@nm.org.

Find articles by Bharat, A. in: JCI | PubMed | Google Scholar

Published November 15, 2024 - More info

Published in Volume 134, Issue 22 on November 15, 2024
J Clin Invest. 2024;134(22):e179527. https://doi.org/10.1172/JCI179527.
© 2024 Liu et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published November 15, 2024 - Version history
Received: January 19, 2024; Accepted: September 26, 2024 View PDF Abstract

A major limitation of immunotherapy is the development of resistance resulting from cancer-mediated inhibition of host lymphocytes. Cancer cells release CCL2 to recruit classical monocytes expressing its receptor CCR2 for the promotion of metastasis and resistance to immunosurveillance. In the circulation, some CCR2-expressing classical monocytes lose CCR2 and differentiate into intravascular nonclassical monocytes that have anticancer properties but are unable to access extravascular tumor sites. We found that in mice and humans, an ontogenetically distinct subset of naturally underrepresented CCR2-expressing nonclassical monocytes was expanded during inflammatory states such as organ transplant and COVID-19 infection. These cells could be induced during health by treatment of classical monocytes with small-molecule activators of NOD2. The presence of CCR2 enabled these inducible nonclassical monocytes to infiltrate both intra- and extravascular metastatic sites of melanoma, lung, breast, and colon cancer in murine models, and they reversed the increased susceptibility of Nod2–/– mutant mice to cancer metastasis. Within the tumor colonies, CCR2+ nonclassical monocytes secreted CCL6 to recruit NK cells that mediated tumor regression, independent of T and B lymphocytes. Hence, pharmacological induction of CCR2+ nonclassical monocytes might be useful for immunotherapy-resistant cancers.

Graphical Abstract Introduction

Tumors augment monopoiesis and orchestrate the epigenetic reprogramming of CCR2+ classical monocytes (CMs) into tumor-associated macrophages that drive immune suppression to promote tumor progression, metastasis, and resistance to immunotherapy (1–3). Monocytes are recruited to the tumor microenvironment via the CCR2/CCL2 axis, which has emerged as a potential target in approaches to slow cancer progression (4, 5).

Some CMs, designated CD14++CD16– in humans and LY6Chi in mice, progressively differentiate into intermediate monocytes (IntMs; CD14++CD16+ in humans and LY6Cint in mice) and nonclassical monocytes (NCMs; CD14+CD16++ in humans and LY6Clo in mice) (6, 7). Under steady-state conditions, this process is governed by the transcription factors C/EBPβ and NR4A1, as evidenced by the monocytopenia of NCMs in Cebpb–/– or Nr4a1–/– mice (8–11). During homeostasis, these naturally occurring NCMs (N-NCMs), identified as CX3CR1hiCCR2–MHC IIlo by use of flow cytometry, patrol the vasculature, engaging with circulating tumor cells to mitigate metastasis (11). Nevertheless, their migration to extravascular tumors is limited due to the absence of CCR2 (8).

LY6Cint IntM cells, the immediate precursors of NCMs, are heterogeneous. Other studies have shown that cells within this subset demonstrate elevated CCR2, MHC II, and CD209 expression, distinguishing them both ontogenetically and phylogenetically from another cluster that is CCR2–MHC II– and gives rise to NR4A1-dependent LY6Clo N-NCMs (9). This heterogeneity, coupled with observations that the small proportion of remaining LY6Clo NCMs in Nr4a1–/– mice (8) have increased CCR2 and MHC II expression, suggests an alternate NR4A1-independent pathway for the development of LY6Clo NCMs. Muramyl dipeptide (MDP), an agonist of the pattern recognition receptor nucleotide-binding oligomerization domain–containing 2 (NOD2), increases the abundance of LY6Clo NCMs in Nr4a1–/– mice (12); and infection from SARS-CoV-2 virus, which has single-stranded RNA (ssRNA), a natural NOD2 agonist, is associated with the development of a unique NCM subtype with C1q expression in humans. Accordingly, we hypothesized that NR4A1 and NOD2 are necessary for mutually exclusive and transcriptionally dichotomous pathways for NCM differentiation. Our data indicate that during homeostasis, NR4A1 is dominant, driving the differentiation of N-NCMs with canonical CX3CR1hiCCR2–MHC IIlo markers. However, NOD2 activation promoted differentiation of CMs toward an inducible NCM (I-NCM) phenotype with noncanonical CX3CR1loCCR2+MHC IIhi markers. We found that these populations of NCMs performed distinct functions. NOD2-induced I-NCMs inhibited tumor seeding and induced regression of various tumor colonies more robustly than did N-NCMs. We show that unlike N-NCMs, I-NCMs migrated into both vascular and extravascular tumor microenvironments via the CCR2/CCL2 axis, where they released CCL6 to recruit NK cells that promote tumor lysis, independent of T and B lymphocytes. Collectively, our findings suggest that the induction of I-NCMs via NOD2 activation can attenuate tumor metastasis independent of conventional immune pathways necessary for immunotherapies.

Results

Inducible Nod2-dependent NCMs are ontogenetically distinct but are underrepresented in healthy individuals

Ontogenetic dichotomy of inducible NCMs and N-NCMs. We isolated circulating LY6Clo NCMs from Nr4a1–/– mice (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI179527DS1) and found that they had a larger percentage of NCMs expressing MHC II (I-A/I-E), CD14, and CCR2 (Supplemental Figure 1B), and a lower percentage of NCMs expressing CX3CR1 (Supplemental Figure 1C) and PD-L1 (CD274) (Supplemental Figure 1D), when compared with NCMs from WT mice. Induction of sterile inflammation by LPS treatment (Supplemental Figure 1, F–K) and lung or spleen transplantation (Supplemental Figure 2, A–G) resulted in a spontaneous increase in (recipient-origin) LY6Clo NCMs in Nr4a1–/– mice (Supplemental Figure 1, F, G, I, and J, and Supplemental Figure 2, A–E), enabling phenotypic analysis by flow cytometry (Supplemental Figure 1, H and K) and transcriptional analysis by RNA sequencing (RNA-Seq) (Supplemental Figure 2, F and G); this confirmed high expression of Ccr2 and MHC II genes, reminiscent of the NCM population previously reported in resting Nr4a1–/– mice (8) and those induced by the NOD2 agonist MDP (12). We hypothesized that these LY6CloCX3CR1loCCR2hiMHC IIhi NCMs were NOD2-dependent (referred to as inducible NCMs [I-NCMs]) and distinct from the naturally abundant NR4A1-dependent LY6CloCX3CR1hiCCR2loMHC II– NCMs (N-NCMs). Treatment with MDP, but not PBS or MDP L-L isomer control (Figure 1A), induced conversion of LY6Chi CMs into I-NCMs in a dose- (Figure 1B) and time-dependent (Figure 1C) manner in Nr4a1–/– and WT but not in Nod2–/– or Nod2–/– Nr4a1–/– mice (Figure 1A). In contrast, while Nr4a1–/– mice lacked N-NCMs, these were abundant in Nod2–/– mice (Supplemental Figure 1, A and E). Both DAPT, a γ-secretase inhibitor, and gliotoxin, a potent NOTCH2 transactivation inhibitor, reduced N-NCM levels in Nod2–/– mice (Figure 1D) but did not prevent MDP-induced development of I-NCMs in Nr4a1–/– mice (Figure 1E). Cell-intrinsic synergistic NOTCH2 and TLR7 activation can promote the development of LY6Clo NCMs from LY6Chi CMs under inflammatory conditions (13). However, we found that MDP treatment was equally effective in converting LY6Chi CMs into I-NCMs in Ccr2cre Tlr7fl/fl mice (Figure 1F). Additionally, R848, a TLR7/8 agonist, did not promote the conversion of CMs into I-NCMs in Nr4a1–/– and Nod2–/– Nr4a1–/– mice (Figure 1G). The spleen serves as a monocyte reservoir (14, 15), and DLL1, a NOTCH2 ligand and a known promoter of N-NCM development (16), is highly expressed in the marginal zone of the spleen. Hence, we tested whether the presence of the spleen was needed for the development of I-NCMs and N-NCMs. We found that MDP treatment of splenectomized Nr4a1–/– mice (Figure 1, H–J) did not alter induction of I-NCMs in the bone marrow, blood, and lungs. Similarly, in WT mice, splenectomy did not affect N-NCMs for up to 32 days (Figure 1K). Additionally, splenectomy combined with MDP treatment did not affect N-NCMs in Nod2–/– mice (Figure 1L). Hence, we conclude that the spleen is dispensable for the spontaneous development of N-NCMs as well as pharmacological induction of I-NCMs.

Figure 1

Ontogenetic dichotomy of I-NCMs and N-NCMs. (A–C) NOD2 activation in vivo by MDP but not isomer control induced conversion of blood CMs in WT and Nr4a1–/–, but not Nod2–/– or double-mutant Nr4a1–/– Nod2–/–, mice to I-NCMs (A), in a dose- (B) and time-dependent (C) manner. (D and E) Inhibition of the NOTCH2 signaling pathway by either gliotoxin (10 mg/kg) or DAPT (25 mg/kg) did not impair the development of I-NCMs in Nr4a1–/– mice but reduced N-NCMs in Nod2–/– mice. (F and G) Development of I-NCMs was not altered by conditional genetic depletion of TLR7 in CCR2+ CM or TLR7/8 activation by R848 treatment (50 μg/mouse). (H–L) Four weeks after splenectomy or sham surgery, mice were treated with MDP or control for 36 hours, and the monocyte subsets were analyzed by flow cytometry. (H–J) The increase in I-NCMs in the bone marrow (H), blood (I), and lung (J) was not impaired by splenectomy (Splx) in Nr4a1–/– mice. Interstitial macrophages (IMs); monocyte-derived dendritic cells (MoDCs). (K) The ratio of LY6Clo NCMs in blood monocytes was stabilized 2 weeks after splenectomy in the blood of WT B6 mice. n = 5. (L) Ratio of monocyte subsets in total monocytes in BM, blood, and lung in Nod2–/– mice (n = 3–4). Unless otherwise mentioned, MDP treatment indicates 1–2 doses of 10 mg/kg MDP via retro-orbital injection for 36 hours; n = 3–12 mice in each group; data are presented as mean ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001; 2-tailed t test was used for A, F, and G; Kruskal-Wallis (nonparametric) test for C: IntM and NCM, J: NCM, and K: IntM; 1-way ANOVA for the rest of the panels.

I-NCMs and N-NCMs are transcriptionally and phenotypically distinct. To obtain pure cell populations of N-NCMs and I-NCMs, we sorted N-NCMs from Nod2–/– mice and I-NCMs from MDP-treated Nr4a1–/– mice and compared their ultrastructure using transmission electron microscopy (TEM). Qualitatively, the N-NCMs were smaller than the I-NCMs (Figure 2, A–C). Furthermore, I-NCMs had abundant pseudopods, mitochondria, liposomes, and nuclear euchromatin (Figure 2, B and C) relative to N-NCMs and LY6Chi CMs from Nr4a1–/– mice (Figure 2C). We compared the transcriptomes of I-NCMs and N-NCMs using bulk RNA-Seq of flow-sorted cells (Figure 2D). Pathway enrichment analysis of differentially expressed genes (Figure 2E) revealed “Leukocyte activation/cell adhesion/migration,” “Cellular component size,” and “Cellular amide metabolic process” (Figure 2F) in the I-NCMs, consistent with the analysis of cellular ultrastructure (Figure 2, A–C). Genes with increased expression in I-NCMs relative to N-NCMs included MHC II– or MHC II–related genes (Cd74, H2-Aa, H2-Ab1, H2-DMa, H2-DMb1, and H2-Eb1) (Figure 2G) and migration-related genes (S100a4/6/10/11, Mmp12, Fgfr1, and Fn1) (Figure 2H, left). Genes with increased expression in N-NCMs relative to I-NCMs included monocyte conversion–related transcription factors (Cebpb, Nr4a1, pou2f2, Klf4) and hallmark NCM genes (Cd11a, Cd36, Cd43, Cd274, Cx3cr1). Hallmark genes associated with CMs were reduced to a greater extent in N-NCMs than in I-NCMs (Irf4, Ccr2) (Figure 2H, right). In contrast, we found lower expression of Cebpb and Cx3cr1 in I-NCMs compared with N-NCMs, and expression of the Nr4a1, pou2f2, Klf4, Cd43, Cd274, and Ccr2 genes in I-NCMs was comparable to that in LY6Chi CMs (Figure 2H, middle and right). Upregulation of transcription factors (such as Irf4, Cebpa, Myc, Batf3, Id2, and Id3) (Figure 2H and Supplemental Figure 3A, left) and differentiation-related genes (Supplemental Figure 3A, right) in I-NCMs compared with N-NCMs suggested distinct transcriptional pathways for their differentiation. Moreover, pairwise comparison revealed the expression of dendritic cell–like (Supplemental Figure 3B) or macrophage-like gene signatures (Supplemental Figure 3C) in I-NCMs re