Granulomas are organized aggregates of immune cells defined by the presence of macrophages, with a variety of other features (i.e. necrosis and fibrosis) being more variable (Warren, 1976). The evolved function of the granuloma response is thought to be a protective mechanism by which immune cells sequester a foreign body or pathogen, walling-off the threat (Pagán and Ramakrishnan, 2018). Some pathogens are not successfully eliminated, however, leading to chronic granulomas that persist for months or sometimes even years. New in vivo models are needed to study the complicated mechanisms that coordinate the formation of protective granulomas, in order to understand the events that lead to the successful clearance of pathogens that initiate this response.

We seek to identify environmental pathogens that have immense virulence capacity but are defeated by the innate immune system. Chromobacterium violaceum is one such pathogen that invades host cells and replicates in the intracellular niche, but only causes morbidity and mortality in immunocompromised animals or individuals (Macher, 1982). We discovered that during infection, wildtype (WT) C57BL/6 mice develop necrotic liver granulomas in response to this ubiquitous soil microbe (Harvest et al., 2023; Maltez et al., 2015). As soon as 1 day post-infection (1 DPI), liver microabscesses can be macroscopically visualized. These lesions are composed primarily of neutrophils until approximately 3–5 DPI, when, importantly, monocytes traffic into the area and form a mature granuloma starting at 5 DPI. Once the resulting macrophage zone surrounds the infected lesion, bacterial burdens begin to decrease, suggesting that granuloma macrophages are an important cell type for the clearance of C. violaceum. By 21 DPI, virtually all mice clear the infection and resolve the granuloma pathology, leaving small collagen scars in place of lesions (Harvest et al., 2023). Though we identified neutrophils and macrophages as the key immune players in this model, much remains to be learned about the cellular mechanisms that initiate formation of the granuloma in response to C. violaceum, and what signals instruct immune cells to organize within the granuloma architecture. Indeed, by studying the granuloma response that successfully clears C. violaceum, we hope to identify critical cellular mechanisms that underlie the basic biology of the granuloma response.

Within the granuloma response to C. violaceum, neutrophils and then macrophages migrate and assemble in an organized manner. Cellular movement, or chemotaxis, must be carefully regulated during tissue development, homeostasis, and inflammatory responses (Hughes and Nibbs, 2018). Chemotaxis is controlled by small, secreted proteins called chemokines that signal through transmembrane chemokine receptors. Since their discovery in the 1980s, approximately 50 chemokines are now appreciated for their role in cellular chemotaxis (Zlotnik and Yoshie, 2012). The temporal and spatial expression of chemokines and chemokine receptors dictate cellular trafficking, and dysregulation of these systems is linked to many diseases (Turner et al., 2014).

As more chemokines have been identified, there have been multiple revisions to their nomenclature, and now a systematic naming of chemokines and their receptors is in wide use. Chemokines have conserved cysteine residues, and the current naming system categorizes four subfamilies based on the arrangement of these N-terminal cysteines: CXC, CC, XC, and CX₃C (Zlotnik and Yoshie, 2000). Though there are exceptions, most chemokines fit into one of these four subfamilies. Similarly, chemokine receptors fall into four subfamilies based on their chemokine ligand. The naming scheme has become complicated due to promiscuous ligand–receptor interactions, reassigning of mouse and human homologs after syntenic analysis, and divergent evolution of ligands in mice and humans (Nomiyama et al., 2013). Nonetheless, the detailed description of many chemokines and their receptors has been accomplished in both species. Herein, we focus on the mouse chemokines and their role in the C. violaceum-induced murine granuloma.

Inflammatory chemokines are those that are rapidly upregulated in the presence of infection or other inflammatory stimuli (David and Kubes, 2019). Several cell types can upregulate chemokines, creating a gradient of ligand that diffuses away from the point of infection. Still other cell types can respond to this gradient if/when they express the appropriate receptor. Furthermore, activated cells that migrate to the area can also upregulate expression of chemokines, creating a feed-forward loop to enhance cell recruitment. In addition to mediating chemotaxis, chemokines can induce a variety of other cellular responses including proliferation, oxidative burst, and even degranulation (Hughes and Nibbs, 2018). Lastly, it is now appreciated that chemokines also contribute to wound healing and resolution of inflammation, with coordinated efforts between neutrophils and macrophages to clean up debris and halt immune cell infiltration (Soehnlein and Lindbom, 2010).

Here, we use spatial transcriptomics to identify key genes that are upregulated in response to C. violaceum, and assess the importance of CCR2-dependent monocyte trafficking to the site of infection in the liver.

Here, we demonstrate that macrophages are essential for clearance of C. violaceum from the infected liver, and for protection against dissemination into the spleen. Loss of CCR2-dependent monocyte trafficking results in a loss of bacterial containment, ultimately leading to uncontrolled bacterial replication in the liver, evidenced by elevated CFU burdens and increased lesion size.

There are many questions that still remain about the individual and coordinated efforts of neutrophils and macrophages during infection with C. violaceum. It is likely that the tissue-resident Kupffer cells and infected hepatocytes are the first cells to sound the alarm, calling for neutrophils. The initial recruitment of neutrophils likely involves chemokines (i.e. CXCL1 and CXCL2) redundantly with other chemoattractants such as formylated peptides and leukotrienes. However, these neutrophils are unable to clear the infection despite being recruited in large numbers.

Based on our data, CCR2 is an essential chemokine receptor for monocyte trafficking in response to C. violaceum, but we have not yet determined which ligand(s) mediate this response. CCL2 and CCL7 can both bind to CCR2 to induce monocyte trafficking. Importantly, pro-inflammatory cytokines and PAMPs can induce CCL2 expression by most cell types (Shi and Pamer, 2011). In agreement, we see upregulation of Ccl2 in several clusters and deposition of CCL2 protein in wide areas around granulomas, further suggesting that CCL2 may be a critical chemokine that promotes monocyte recruitment in response to C. violaceum. In contrast, Ccl7 is expressed by fewer clusters, and to a lesser degree, and its expression is slightly delayed compared to Ccl2. Deletion of either ligand partially diminished monocyte trafficking in response to Listeria monocytogenes infection, but the individual role of each ligand was unclear (Jia et al., 2008). Future studies using C. violaceum could further elucidate the unique or redundant roles of CCL2 and CCL7. Lastly, adoptive transfer experiments in the context of Listeria infection showed that Ccr2^–/– monocytes are still able to traffic to the site of infection in the spleen (Serbina and Pamer, 2006) and liver (Shi et al., 2010). During C. violaceum infection, we have not yet determined whether CCR2 is required for migration once monocytes have left the bone marrow, as CCR2 is required for this initial egress. We saw a subtle increase in the number of macrophages in the liver of infected Ccr2^–/– mice. Though macrophage numbers in Ccr2^–/– tissues remain considerably lower than seen in WT mice, there are two explanations for the subtle increase: (1) loss of CCR2 may not completely abrogate monocyte recruitment, as monocytes could be migrating via other chemokine receptors, or (2) tissue-resident macrophages, or even tissue-resident hematopoietic stem cells, could undergo emergency hematopoiesis and proliferate in response to infection (Boettcher and Manz, 2017). More studies are needed to assess the origin of this small population of macrophages in Ccr2^–/– mice. Regardless, this small population of macrophages is not sufficient to protect against infection with C. violaceum.

In other granuloma models, the role of CCR2 is less clear. Loss of CCR2-dependent monocyte trafficking enhances clearance of Y. pseudotuberculosis (Zhang et al., 2018), which is a surprising result as typically macrophages would be expected to be important to clear infections. The role of CCR2 during M. tuberculosis infection is strain-dependent, and also varies depending on the dose and route of infection (Dunlap et al., 2018; Peters et al., 2001; Scott and Flynn, 2002). Though there are similarities between these infection models and C. violaceum, there are numerous differences. For example, expression of specific chemokines in response to M. tuberculosis differs from those we observe in response to C. violaceum, especially chemokines that attract T cells (Kang et al., 2011). A key concept in the M. tuberculosis field is that a delicate balance exists between cellular recruitment to control infection, and excess inflammation that causes disease symptoms (Monin and Khader, 2014). Furthermore, excess recruitment of monocytes to M. tuberculosis-induced granulomas leads to increased bacterial replication due to the ability of M. tuberculosis to inhibit degradation within phagosomes in which it resides (Domingo-Gonzalez et al., 2016; Slight and Khader, 2013). In contrast, C. violaceum appears to lack sufficient virulence factors to enable it to replicate within macrophages (Batista and da Silva Neto, 2017). Importantly, while M. tuberculosis bacterial burdens plateau at 21 DPI, almost all mice clear C. violaceum by this timepoint. Though decades of research have been dedicated to investigating M. tuberculosis, fewer studies involving other granuloma-inducing pathogens have been performed. As we continue to study the cellular mechanisms that allow for successful granuloma formation and clearance of C. violaceum, it will be interesting to compare the two pathogens, as future studies could shed light on key differences that result in successful pathogen clearance.

In WT mice, neutrophil recruitment wanes as the granuloma matures, which coincides with clearance of C. violaceum. However, in the Ccr2^–/– mice, we see elevated neutrophil numbers at 5 DPI, suggesting that neutrophils are continuously recruited in the absence of macrophages. Under normal circumstances, endocytosis of chemokines by endothelial cells helps to diminish chemokine gradients, limiting prolonged neutrophil recruitment (Kolaczkowska and Kubes, 2013). Future studies could investigate the various signals that diminish neutrophil recruitment in WT mice during clearance, and why this fails in Ccr2^–/– mice. Another interesting component of the granuloma response is the spatial arrangement of neutrophils and macrophages within the granuloma. In vitro studies found that CCR1 and CCR5 differentially affected monocyte localization within a transwell system, implying that a system exists for fine-tuning the exact location of macrophages within inflamed tissues (Shi and Pamer, 2011). These receptors are highly upregulated in the C. violaceum-induced granuloma and are thus good candidates for balancing the localization of macrophages between the coagulative necrosis zone and healthy tissue outside the granuloma.

Lastly, this dataset inspires a number of new hypotheses related to granuloma resolution and tissue repair after bacterial clearance. Chemokines undergo a variety of post-translational modifications, such as glycosylation, nitration, citrullination, and proteolytic cleavage, which can either enhance or abrogate their activity (Vanheule et al., 2018). For example, nitration of CCL2 and CCL3 by peroxynitrite was shown to reduce monocyte and neutrophil chemotaxis, respectively (Sato et al., 1999; Sato et al., 2000). Furthermore, binding to atypical receptors can also affect chemokine availability, representing another mechanism to resolve inflammation (Hansell et al., 2006; Ulvmar et al., 2011). Of particular interest is the implication of matrix metalloproteinases (MMPs) in regulating chemokine functions. MMPs can not only directly cleave chemokines, they can also cleave various chemokine-binding proteins that help establish the chemokine gradient (Parks et al., 2004). Several studies have found that MMPs can cleave chemokines to alter their function, either increasing or decreasing their receptor binding activity. For example, MMP-2 cleaves both CXCL12 and CCL7, abolishing their ability to induce chemotaxis (McQuibban et al., 2000; McQuibban et al., 2001); importantly, all three of these genes are upregulated during C. violaceum infection (Table 1, Table 3, Table 4). Furthermore, MMP-2 and MMP-9 have been extensively studied in the context of lung inflammation, both of which are important to limit tissue damage (Greenlee et al., 2006). MMP-9 has also been shown to promote or inhibit liver fibrosis and wound repair, depending on the context (Feng et al., 2018). An unsolved mystery during infection with C. violaceum is how the chemotaxis of neutrophils and monocytes is abrogated when the infection is cleared, and how wound repair and resolution is initiated. Future studies could characterize the role of MMPs during resolution, especially MMP-9 and its various targets in relation to wound repair.

Analysis of a spatial transcriptomics dataset revealed the upregulation of many chemokines and their receptors during murine infection with C. violaceum. Here, we show that CCR2 is an essential chemokine receptor for monocyte trafficking, which enables the formation of mature granulomas with organized macrophage zones. Importantly, loss of organized macrophages leads to loss of bacterial containment. This work has given new insight into the function of chemokines during granuloma formation, and this model of C. violaceum-induced granuloma formation will be useful in exploring the unique and redundant roles of chemokines during infection.

All study data are included in the article as source data files, and the spatial transcriptomics dataset is deposited with Dryad (https://doi.org/10.5061/dryad.zpc866thz). Streamlined RStudio code is included in Source code 1.

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   (2024) Dryad Digital Repository

   Spatial transcriptomics of an innate granuloma in a mouse infection model with Chromobacterium violaceum.

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Author details

1. Megan E Amason

   1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, United States
   2. Department of Ophthalmology, Duke University School of Medicine, Durham, United States
   3. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, United States
   4. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, United States
   5. Department of Pathology, Duke University School of Medicine, Durham, United States

   Contribution

   Data curation, Formal analysis, Software, Conceptualization, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0000-2834-5834

2. Cole J Beatty

   1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, United States
   2. Department of Cell Biology, Duke University School of Medicine, Durham, United States

   Contribution

   Formal analysis, Software, Conceptualization

   Competing interests

   No competing interests declared

3. Carissa K Harvest

   1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, United States
   2. Department of Ophthalmology, Duke University School of Medicine, Durham, United States
   3. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, United States
   4. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, United States
   5. Department of Pathology, Duke University School of Medicine, Durham, United States

   Contribution

   Formal analysis, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

4. Daniel R Saban

   1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, United States
   2. Department of Cell Biology, Duke University School of Medicine, Durham, United States

   Contribution

   Supervision, Project administration

   Competing interests

   No competing interests declared

5. Edward A Miao

   1. Department of Integrative Immunobiology, Duke University School of Medicine, Durham, United States
   2. Department of Ophthalmology, Duke University School of Medicine, Durham, United States
   3. Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, United States
   4. Department of Pathology, Duke University School of Medicine, Durham, United States

   Contribution

   Data curation, Supervision, Funding acquisition, Writing – review and editing

   For correspondence

   edward.miao@duke.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7295-3490

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