Differential requirement of Formyl Peptide Receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

Abstract

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. This study investigates the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed using a TB model of hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion showed no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1 ^-/- mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased bacterial persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1 ^-/- neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

Keywords: Formyl peptide receptors; G-protein coupled receptors; Host defense; Immunity; Mycobacterium tuberculosis.

Figure 1.. Fpr1 and 2 expression in the lungs is associated with TB disease.

(a) Wild type (Wt) and Il1r1^−/− mice were infected via aerosol with Mtb HN878 smyc’:: mCherry (Mtb), delivering approximately 100 CFU to the lungs. At 26 dpi, Fpr1 (a) and Fpr2 (b) expressions were quantified in the lungs using qPCR. Expression levels in Il1r1^−/− mice were calculated relative to those in Wt C57BL/6 (Wt) mice. (c, d) Representative immunofluorescence images of formalin-fixed paraffin-embedded (FFPE) lung sections from Mtb-infected Wt and Il1r1^−/− mice. Left panels: DAPI (blue) stains nuclei, Fpr1 (red). Right panels: DAPI (blue), Fpr2 (yellow). Quantification of Fpr1 and Fpr2 expressions in lung sections expressed as corrected total fluorescence intensity (CTCF) from three fields of view per group, representing one of two experiments. Data represent n=3 samples per time point. Error bars show Mean ± SEM. Statistical analysis was performed using an unpaired t-test. *P<0.05, ****P<0.00001. (e) qPCR analysis of FPR1 and FPR2 expressions in rabbit lungs post Mtb infection with strains HN878 and CDC1551 at 3 hours and 4 weeks. (f) Formyl Peptide Receptor (FPR) 1 and 2 expression in human TB before and after anti-TB therapy: RNA-seq data was extracted from publicly available dataset previously published (GSE19435). Data is comprised of whole blood transcriptional signatures obtained from two different cohorts. Data sets were downloaded from NCBI as the Longitudinal TB Treatment in a UK cohort (GSE19435) and (g) whole blood transcriptional signatures in latent TB (LTBI) and active TB in a South African Cohort (GSE19442). Genes were identified based on their Ilumina IDs; FPR1(ILMN_2092118), FPR2 (ILMN_2392569, ILMN_1740875), extracted, plotted and analysed by unpaired student t-test versus the indicated groups. **p<0.01, ***p<0.001 and ****p<0.0001.

Figure 2.. Protective effects of Fpr1 deletion on tuberculosis outcomes in mice.

Wt and Fpr1^−/− mice on a C57BL/6 background were aerosol-infected with Mtb HN878 reporter bacteria as per the protocol in Figure 1. Necropsies and tissue analyses were conducted at 32 dpi, with 4–6 mice per group. (a) Percentage of weight change upto 32 dpi in Mtb-infected Wt and Fpr1^−/− mice. (b) Bacterial load in the lungs and spleens measured in CFU per ml at 32 dpi in both Wt and Fpr1^−/− mice. (c) Flow cytometry assessment of myeloid cells, including neutrophils, macrophages, and monocytes at 32 dpi in both Wt and Fpr1^−/− mice. (d) Flow cytometry assessment of infected neutrophils and macrophages at 32 dpi in both Wt and Fpr1^−/− mice. Live infected neutrophils were marked with viability dye-CD11b+Ly6G+smyc’::mCherry+, while dead or dying neutrophils were identified using viability dye+CD11b+Ly6G+smyc’::mCherry+. Live infected macrophages were marked with viability dye-CD11b+Ly6G-CD11c+MHCII+SiglecF-smyc’::mCherry+. (e) Flow cytometry for T-lymphocytes (CD4+, CD8+) and B lymphocytes (CD19+) at 32 dpi in Wt and Fpr1^−/− mice. (f) Quantification of cytokines (IL-1α, IL-1β, IL-6, TNF-α) in lung homogenates from both Wt and Fpr1^−/− mice at 35 dpi. n=3 per group. (g) Lung Histopathology: Images and blind scoring of inflammatory lesions in the lungs of Wt and Fpr1^−/− mice at 35 dpi. n=4–5 mice per group. Error bars represent Mean ± SEM. Statistical analysis involved a two-way ANOVA for panel (a), with significance determined by Tukey’s multiple comparison test (****p<0.0001). Unpaired t-tests were conducted for panels (b-g). *P<0.05, ***p<0.001, and “ns” denotes non-significant results.

Figure 3.. Blocking Fpr1 in susceptible mice increased bacterial growth in the lungs.

(a) Experimental setup: Il1r1^−/− mice were exposed to an aerosol containing approximately 100 CFU of Mtb HN878 reporter bacteria. Starting one day before infection (day −1), the mice were given either a vehicle or Fpr1 inhibitors every other day. The Fpr1 inhibitors used were a combination of Cyclosporin H (4 mg/kg) and HCH6–1 (4 mg/kg), administered orally. Measurements were taken on days 14, 21, and 25 post-infection. (b) Bacterial load in the lungs was determined by CFU counts. (c) Flow cytometry was used to assess Mtb-infected neutrophils (live on the left), (d) dead/dying on the middle and (e) macrophages in the right. (f) Lung Histopathology: Representative images of H&E-stained FFPE lung sections are shown for the specified infection times. (g) Quantification of necrotic lesion areas illustrates the progression of disease over time in both vehicle-treated and inhibitor-treated mouse lungs. Data are from n=3–7 mice per group. Results from day 25 are combined from two separate experiments. Error bars represent the mean ± SEM. Statistical significance was assessed using an unpaired t-test compared to respective controls. *p<0.05; ****p<0.0001; ‘ns’ denotes non-significant results.

Figure 4.. Fpr1 blockade impairs bacterial control in immunocompetent C3HeB mice.

(a) Experimental design: C3HeB mice were infected with Mtb HN878 reporter bacteria via aerosol and treated with either a vehicle or Fpr1 inhibitors according to the schematic. Evaluations were conducted at 14- and 35 dpi. (b) Bacterial burden in the lungs and spleens of both vehicle-treated and inhibitor-treated mice was measured and expressed as colony-forming units (CFU). (c) Flow cytometry was used to assess Mtb-infected neutrophils (live on the left); (d) dead/dying on the middle) and (e) macrophages (right panel) at the specified time points post-infection. (f) Representative histopathology images of H&E-stained FFPE lung sections. (g) Quantification of necrotic lesion areas in the lungs at 14 and 35 dpi for both vehicle- and inhibitor-treated mice. n = 4 per group. Error bars represent Mean ± SEM. Statistical significance was assessed using an unpaired t-test compared to respective controls. *p<0.05; ***p<0.001; ‘ns’ denotes a non-significant result.

Figure 5.. Fpr1 plays a contrasting role in macrophages and neutrophils during Mtb infection.

(a) Experimental setup: Bone marrow-derived neutrophils from Wt and Fpr1^−/− mice were isolated using magnetic cell sorting (MACS) and infected with Mtb HN878 at a multiplicity of infection (MOI) of 3.0 for 4 hours. After removing extracellular bacteria through washing, cells were further incubated for 24 hours. The intracellular bacterial load was then assessed using CFU analysis. (b) Intracellular bacterial load in neutrophils is presented as CFU counts. (c) Experimental setup: Bone marrow-derived neutrophils from Wt, Pad4^−/−, and Cybb^−/− mice were isolated using MACS and infected with Mtb HN878 at an MOI of 3.0, as described in (a), with or without the addition of 100nM fMLP. (d). Bacterial burden in these neutrophils was measured at 24 hours post-infection and is shown as CFU. (e) Experimental setup: Bone marrow-derived macrophages (BMDMs) from Wt and Fpr1^−/− mice were infected with Mtb HN878 at an MOI of 3.0. (f) Bacterial load in BMDMs was determined at various time points post-infection and expressed as CFU counts. The experiments were conducted with n=3 replicates per group and are representative of two independent experiments. Error bars represent Mean ± SEM. Statistical analysis was performed using unpaired t-tests. *p<0.05; ‘ns’ denotes a non-significant result.

Figure 6:. Model depicting the differential roles of Fpr1 in myeloid cell anti-mycobacterial functions.

This model illustrates the contrasting effects of Fpr1 expression in neutrophils and macrophages during Mtb infection. Fpr1 expression in neutrophils is essential for effectively controlling Mtb growth within these cells. Conversely, in macrophages, Fpr1 expression impedes their ability to control intracellular Mtb growth, as evidenced by a reduced bacterial burden in macrophages lacking Fpr1. This model highlights the previously unrecognized importance of Fpr1 in the immune response of myeloid cells against tuberculosis. Graphic was designed by www.biorender.com.