Despite effective drug therapy, tuberculosis (TB) remains a leading cause of death globally. Annually, approximately 10 million fall ill, and there were 1.6 million deaths in 2021 (1). The causative agent of human TB, Mycobacterium tuberculosis (Mtb), is an ancient pathogen that primarily affects the lungs, although dissemination to extrapulmonary sites is possible (2–5). Protective TB immunity depends on the vigorous induction of T cells and innate effectors such as macrophages and neutrophils (6). However, chronic inflammation due to persistent Mtb infection is a key driver of immunopathology manifesting as cavitary disease in TB patients (7, 8). Cavities facilitate the expulsion of aerosolized bacilli via the airways for transmission to a new host, thereby completing the bacterial life cycle. A considerable body of evidence indicates that negative immune regulation of the T cell response to Mtb is critical to preventing excessive inflammation and maintaining lung health during Mtb infection (9).

Immune checkpoint (IC) molecules are inhibitory receptors that induce immunosuppressive signaling pathways (10). Primarily expressed by T cells, ICs play a key role in maintaining self-tolerance and limiting immunopathology. One of the most well-studied of these is programmed cell death protein 1 (PD-1), primarily due to its exploitation in novel cancer therapies. Inhibition of PD-1 or its receptors results in more robust Th1 responses and has proven a successful therapeutic approach to treating multiple cancers (11). However, numerous clinical case reports and pharmacovigilance analyses have shown that blockade of PD-1 can lead to reactivation of latent TB (9, 12). This is mainly thought to be due to a reinvigorated T-cell immune responses driving immunopathology and progression of previously stable TB lesions (13–15). Likewise, PD-1 deficiency in mice significantly exacerbated Mtb infection and was rapidly lethal (16, 17). Therefore, unrestrained T cell responses appear to undermine effective TB immunity.

Monocytes are recruited from the blood at the site of Mtb infection and can differentiate into dendritic cells and macrophages (18). In mouse models, dendritic cells and macrophages are the primary sites of Mtb replication during later stages of infection (19). Importantly, Mtb has been shown to modulate monocyte surface markers (20), and several of these, such as CD64, CD163, and CD14, have been identified as putative biomarkers of active TB disease (21). Resident alveolar macrophages (AMs) constitute the lung’s first line of immune cell defense against Mtb (22). To limit harmful immune responses that could impair lung function, AMs maintain an inherently anti-inflammatory phenotype characterized by constitutive expression of inhibitory molecules, including the CD200 receptor (CD200R) (23, 24). In the airway, the interaction between CD200R on AMs and CD200 expressed by alveolar epithelial cells induces a hyporesponsive phenotype (23). Mice deficient for the inhibitory CD200-CD200R pathway are highly susceptible to influenza and F. tularensis infection due to excessive inflammation (25, 26). Conversely, engagement of the CD200-CD200R axis mitigates lung inflammation in asthma (27). Patients suffering from the systemic inflammatory syndrome sarcoidosis exhibited decreased expression of CD200R on circulating monocytes compared to healthy controls (28). In light of these observations, we questioned whether the CD200-CD200R axis played a role in the immune response to Mtb infection.

To determine CD200R expression, we analyzed PBMCs from participants with active TB (ATB) and healthy controls (HC) by flow cytometry ( Supplementary Figure 1 ). ATB was associated with significantly lower frequency of CD200R-expressing CD14⁺ monocytes (median = 13.1%) compared to HC (median = 31.2%, P = <0.0001; Figure 1A ). This is consistent with an enhanced pro-inflammatory phenotype of circulating monocytes from TB patients (35). In contrast, there were no differences in the frequency of CD200R-expressing CD3⁺ T cells in ATB (median = 57.20%) compared to HC (median = 57.7%, P = 0.78) ( Figure 1B ).

Next, to determine whether CD200R expression was also reduced on myeloid cells in the lung, we performed flow cytometry on lung tissue sections isolated from 3 individuals (3 tissue samples per patient) with TB disease undergoing surgical resection to treat TB sequelae (31). The 3 tissue blocks were selected by the operating surgeon based to include the most and least diseased sections of the lung tissue removed. It is important to note, however, that this is a crude categorization as the extent of disease varies between subjects and the selection of tissue blocks is based on macroscopic appearance. All tissue blocks were homogenized to generate a single cell suspension and CD200R expression determined by flow cytometry as above. As CD14⁺ monocytes typically mature into macrophages or other myeloid subsets when they migrate into inflamed tissue, CD200R expression levels were assessed on CD14⁺ monocytes and CD11b⁺HLA-DR⁺ cells, consistent with a macrophage phenotype. Figure 1C shows CD200R expression on two tissue blocks from a TB-diseased lung corresponding to the most and least diseased tissue sections as defined by the surgeon. In the more diseased tissue block, CD200R is expressed on CD3⁺ T-cells but only on a very small fraction (<10%) of CD14⁺ monocytes or CD11b⁺HLA-DR⁺ myeloid cells ( Figure 1C ). In contrast, in the less diseased tissue block from the same lung, most CD14⁺ and CD11b⁺HLA-DR⁺ cells expressed CD200R. This pattern of expression was reflected in one of the two other lungs studied (bright green dots), while a third lung (red dots) had a similar frequency of CD200R-expressing myeloid cells in all tissue blocks analyzed ( Figure 1D ). Moreover, in two of the lungs, CD200R expression on CD11b⁺HLA-DR⁺ cells as well as CD14⁺ cells were markedly increased in sections exhibiting less pathology ( Figure 1E ). These data suggest that CD200R expression on myeloid cells is heterogenous in TB diseased lung tissue and tends to be lower in tissue sections with more overt disease.

To investigate this further, we next determined the relationship between CD200R and TB lesions within the lung by fluorescence microscopy. A lung tissue section containing multiple TB lesions was scanned and analyzed using the TissueFAX software to extract single-cell expression data. The cellular composition of granuloma displayed a conventional CD68⁺ macrophage cuff around an acellular core, surrounded by an outer zone of CD3⁺ T cells ( Figure 2A ). In contrast to the circulating monocytes ( Figure 1A ), CD200R was highly expressed on CD68⁺ cells located on the periphery of granulomas ( Figure 2A ). As shown in Figure 2B , CD200R was expressed on CD68⁺ macrophages both distal and proximal to the granuloma core. As expected, macrophages proximal to the granuloma center (indicated with a red star) displayed a more epithelioid morphology, consistent with granuloma-associated macrophages. Interestingly, CD200R expression was less consistent within these macrophages, particularly near the granuloma core (indicated with a pink star). To examine this further, nine granulomas within the tissue section were scanned, and CD200R expression levels were quantified on CD68⁺ macrophages within each lesion ( Supplementary Figure 2 ). In addition, TissueFAX software allows the measurement of area by demarcation, so granuloma size was determined this way. This revealed a significant negative association between lesion size and macrophages CD200R expression level ( Figure 2C ). Taken together, these data suggest that CD200R is expressed on granuloma-associated macrophages within the lung, but the expression level is variable and may be reduced in cells closest to the granuloma core and in the larger granuloma.

In light of these results, we tested the effect of CD200R inhibition on bacterial control using a 3D granuloma model (33). For this, PBMCs were infected with bioluminescent Mtb and suspended in an alginate-collagen matrix. This mixture was passed through a cell encapsulator to generate microspheres, allowing the cells to aggregate in 3D, with collagen providing an extracellular matrix and thus mimicking the lung tissue environment. Previously, we demonstrated in this model that PD-1 blockade accelerated Mtb growth, driven by an exacerbation of the TNF-α response (36). The addition of 200 μg/mL of CD200R blocking antibody resulted in a significant increase in bacterial growth, as shown by an increase in the luminescent signal at day 7 ( Figure 3A ), previously shown to correlate closely with Mtb burden (37). Addition of 20 μg/mL of anti-CD200R or an isotype control did not affect Mtb growth. In contrast to PD-1 inhibition, which greatly increased TNF-a secretion, CD200R blockade led to a decrease in TNF-a levels, although this did not reach statistical significance ( Figure 3B ). Cell viability was assessed to test whether the increase in Mtb growth upon CD200R blockade was due to cell death. Incubation with either the isotype control or anti-CD200R, however, did not significantly impact cell viability compared to untreated controls ( Figure 3C ). Finally, we examined CD200R gene expression in published data generated by our groups from human TB granuloma and the 3D model (34). Bulk RNA sequence data from laser captured untreated TB granuloma identified in lymph nodes showed a slight reduction in the expression of CD200 and CD200R compared to uninfected lymph nodes, although the difference did not reach statistical significance ( Figure 3D ). Interestingly, we observed a similar decrease in the expression of PDCD1 (gene encoding PD-1) but the expression of the genes encoding its two ligands CD274 (PD-L1) and PDCD1LG2 (PD-L2), were highly increased. In the 3D model, we observed a similar and statistically significant reduction of CD200R1 expression during Mtb infection compared to uninfected spheres, but no change in CD200 expression ( Figure 3D ). As in the lymph nodes, CD274 and PDCD1LG2 were highly increased, consistently with the overall transcriptional similarity between in vivo TB granuloma and the 3D model system (34). It is worth noting that highly elevated PDL1 protein expression has been observed in human TB lung granuloma (38). These data are consistent with reduction in of CD200R expression observed on circulating monocytes in TB patients ( Figure 1A ) and with reduced CD200R expression in progressive lung granuloma ( Figure 2B, C ).

Here, for the first time to our knowledge, we show that CD200R expression is significantly and consistently decreased in circulating CD14⁺ monocytes from TB patients compared to healthy controls. In TB lung tissue, CD200R expression was variable across the diseased lung on CD14⁺ monocytes and CD11b⁺HLA-DR⁺ macrophages, and some tissue sections, obtained from the most diseased parts of the lung, contained little or no CD200R⁺ myeloid cells. Histological analysis revealed that CD200R was expressed on CD68⁺ macrophages associated with granuloma. However, in TB-diseased lungs, CD200R expression levels appeared lower on macrophages closest to the necrotic core. In addition, analysis of all 9 TB lesions in this tissue section suggests that expression levels of CD200R decreased as lesion size increased. Although significant, we are cautious in extrapolating this data, as it represents a single slide from a single participant and includes an outlying data point from only a single larger lesion. It will, therefore, be important to confirm these findings in additional samples. The lack of uninfected controls for the lung homogenates as well as the lung tissue section is a limitation of this study. Nevertheless, it is clear that CD200R expression levels on circulating monocytes are modulated in TB disease, strongly support a potential role for altered CD200R signaling within the lung associating with TB disease. This hypothesis is supported by independent experiments showing that the blockade of CD200R significantly increased Mtb growth in the 3D-granuloma model; and that Mtb infection in the 3D model induces reduced gene expression of CD200R1. Therefore, these data indicate that, like T-cells, regulation of myeloid immune responses in the lung is likely to play an important part in limiting immunopathology in TB.

The majority of TB in adults develops in immune-experienced host and is termed post-primary disease. In post-primary TB, pathology can develop as a function of immunodeficiency, as in the case of progressive HIV or hyperinflammation (39). This later mechanism is exemplified by clinical and experimental studies showing that the loss of negative regulation of T-cells via PD-1/PDL-1 accelerates the onset of TB disease (9, 12, 13). Equivalent data is unavailable for CD200R, but LPS-induced lung inflammation is exacerbated in CD200-deficient rats as a result of greater alveolar macrophage activation (40). Likewise, mice lacking CD200 have an exaggerated response to influenza infection, leading to slower resolution of infection and greater lung damage (25). Interestingly, several viruses have evolved to express CD200-like proteins on the surface of infected cells, which interact with CD200R and inhibit killing (41). Thus, it is not unexpected that the CD200/CD200R axis might play an important role in modulating TB immunopathology. Like PD-1/PDL-1, CD200R has aroused interest as a potential target in the treatment of tumors (42). CD200 is highly expressed on tumor cells, impairs antitumor immune responses, and is associated with poor prognosis. Although the blockade of CD200 has been shown to promote antitumor immunity, definitive clinical trial data on efficacy is still lacking (41). If CD200/CD200R blocking agents proceed to further clinical trials, however, it will be important to look for early signals of TB reactivation. In conclusion, these data show that CD200R expression is modulated during active TB in humans, on both circulating monocytes and myeloid cells within TB-diseased lungs, and is likely to play an important role in balancing the inflammatory responses during Mtb infection.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

The studies involving humans were approved by Biomedical Research Ethics Committee (UKZN). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

MA: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing, Conceptualization. LT: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. NH: Formal Analysis, Methodology, Resources, Software, Writing – review & editing. MC: Data curation, Formal analysis, Methodology, Resources, Software, Writing – review & editing. MR: Writing - review & editing, Formal analysis. KN: Methodology, Resources, Software, Writing – review & editing. HK: Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – review & editing. FK: Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – review & editing. MbH: Methodology, Resources, Supervision, Writing – review & editing. RM: Methodology, Resources, Supervision, Writing – review & editing. MuH: Methodology, Resources, Supervision, Writing – review & editing. MoH: Methodology, Project administration, Resources, Supervision, Writing – review & editing. AS: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. PE: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – review & editing. AL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.