Inflammatory monocytes hinder antiviral B cell responses

Abstract

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. Here we analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of the draining lymph node where they extensively interact with CD11b⁺Ly6C^hi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon-, CCR2-dependent fashion and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment or impairment of their nitric oxide-producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. In conclusion, our results identify inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.

Figure 1. Spatiotemporal dynamics of B cell activation in response to VSV and LCMV infection.

(A) Neutralizing Ab titers in the sera of C57BL/6 mice that were infected s.c. with 10⁵ pfu of VSV (gray) or 10⁵ ffu of LCMV (black). n = 5; results are representative of at least three independent experiments. (B) Neutralizing Ab titers in the sera of D_HLMP2A mice that were transferred with 5 x 10⁶ purified VI10YEN (gray) or KL25 (black) B cells 18h prior to s.c. infection with VSV or LCMV, respectively. n = 5; results are representative of at least two independent experiments. (C) Multiphoton intravital (left and middle panels) or confocal (right panel) micrographs in the popliteal LN of a D_HLMP2A mouse that was injected with VI10YEN (red) and WT (cyan) B cells 18h prior to s.c. VSVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. Dotted white line denotes B cell follicles. Scale bars represent 50μm (left and middle panels) or 100μm (right panel). Results are representative of at least five independent experiments. See also Movie S2. (D) Multiphoton intravital (left and middle panels) or confocal (right panel) micrographs in the popliteal LN of a D_HLMP2A mouse that was injected with KL25 (red) and WT (cyan) B cells 18h prior to s.c. LCMVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. Dotted white line denotes B cell follicles. Scale bars represent 50μm (left and middle panels) or 100μm (right panel). Results are representative of at least five independent experiments. See also Movie S3. (E) Percentage of WT (blue) and VI10YEN (red) B cells within the indicated LN compartments 72h after VSV infection. IFA, Interfollicular area. n = 2; results are representative of at least three independent experiments. See also Movie S2. (F) Percentage of WT (blue) and KL25 (red) B cells within the indicated LN compartments 72h after LCMV infection. IFA, Interfollicular area. n = 2; results are representative of at least three independent experiments. See also Movie S3. (G) Meandering index of WT (blue) or KL25 (red) B cells in the interfollicular area (total, left panel) or in interfollicular areas that were > 15 μm distant from GFP⁺ cells (GFP^- areas, right panel), 72h after LCMV infection. n = 2. Results are representative of at least two independent experiments. See also Movie S3. Results are expressed as mean ± SEM. **p < 0.01, ***p < 0.001.

Figure 2. Inflammatory monocytes, recruited to LNs in a type I interferon- and CCR2-dependent fashion, suppress LCMV-specific Ab responses.

(A) FACS plots showing CD11b and Ly6C expression in the popliteal LNs of mice injected in the footpad with PBS (left) or LCMV (right) 72h earlier. Numbers show the percentage of cells within the gates. Plots are representative of at least ten independent experiments. (B) Number of CD11b⁺Ly6C^hi cells within the popliteal LNs of WT mice, at the indicated time points after s.c. LCMV infection. Results are representative of at least three independent experiments. (C) Number of CD11b⁺Ly6C^hi cells within the popliteal LNs of WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or anti-Gr-1-treated WT mice (green), 72h after s.c. LCMV infection. (D-E) 5 x 10⁶ KL25 B cells were transferred into WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or into WT mice injected with anti-Gr-1 (green) or isotype control (black) Abs prior to s.c. LCMV infection. LCMV-GP-binding (D) or neutralizing (E) Abs were measured in the sera seven days after LCMV infection. LCMV-GP-binding Abs are expressed as fold induction over uninfected controls. Results are representative of at least five independent experiments. (F) LCMV-GP-binding Abs in the sera of WT (black), IFNAR^-/- (red), CCR2^-/- (blue) mice or anti-Gr-1-treated WT mice (green), 7 days after s.c. LCMV infection. Data are expressed as fold induction over uninfected controls. Results are representative of two independent experiments. (G) KL25 B cells were transferred into WT (black), CCR2^-/- (blue) or CCR2^-/- mice reconstituted with 5 x 10⁶ WT monocytes (yellow). LCMV-GP-binding Abs (fold induction over uninfected controls) were measured 7 days after s.c. LCMV infection. Results are representative of at least two independent experiments. (H) LCMV titers 7 days p.i. in popliteal LNs of WT (black) or CCR2^-/- (blue) mice that were transferred with 5 x 10⁶ KL25 B cells prior to s.c. LCMV infection. Results are representative of at least two independent experiments. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3. Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells and impair their survival.

(A) Confocal micrographs in the popliteal LNs of CX3CR1^GFP/+;CCR2^RFP/+ mice that were transferred with fluorescent WT (cyan) and KL25 (red) B cells, injected s.c. with either PBS (left panel) or LCMV (right panel) and sacrificed 72h after infection. Scale bars represent 100μm. Dotted white line denotes B cell follicle. Results are representative of at least three independent experiments. (B) Multiphoton intravital micrograph in the popliteal LNs of a LCMV-infected CX3CR1^GFP/+;CCR2^RFP/+ mouse that was transferred with fluorescent WT (cyan) and KL25 (red) B cells and sacrificed 72h after infection. Scale bar represents 20 μm. Result is representative of at least three independent experiments. See also Movie S4. (C) Quantification of the contact time (minutes) between CXCR1⁺ CCR2⁺ cells and WT (blue) or KL25 (red) B cells in the popliteal LNs of CX3CR1^GFP/+;CCR2^RFP/+ mice that were infected s.c. with LCMV 72h earlier. See also Movie S4. n = 4. Results are representative of at least 2 independent experiments. (D-E) 5 x 10⁶ KL25 B cells were transferred into WT (black), IFNAR^-/- (red), CCR2^-/- (blue) or WT mice injected with anti-Gr-1 (green) or isotype control (black) Abs and infected s.c. with LCMV. Frequency of annexin V⁺ apoptotic KL25 B cells (D) and number of KL25 B cells (E) were quantified in the popliteal LNs 3 days after infection. Results are representative of at least five independent experiments. (F) 5 x 10⁶ VI10YEN B cells were transferred into WT mice prior to s.c. immunization with PFA-inactivated VSV, and either simultaneously infected (red) or not (black) with LCMV. VI10YEN B cells were quantified in the popliteal LN 72h after immunization. Results are representative of at least two independent experiments. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Inflammatory monocytes inhibit B cell responses via nitric oxide.

Irradiated WT recipients were reconstituted with WT (black), CCR2^-/- (blue), a 1:1 mixture of WT and CCR2^-/- (gray) and a 1:1 mixture of iNOS^-/- and CCR2^-/- (orange) bone marrow 8 weeks prior to injection with 5 x 10⁶ KL25 B cells followed by s.c. LCMV infection. (A) Number of CD11b⁺Ly6C^hi cells recovered in the popliteal LNs 3 days after infection. (B) Number of KL25 B cells recovered in the popliteal LNs 3 days after infection. (C) Serum LCMV-GP-binding Abs (fold induction over uninfected controls) measured 7 days after infection. Data are representative of at least two independent experiments. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.