MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2.

Humoral immune responses initiate in the lymph node draining the site of viral infection (dLN). Some viruses subvert LN B cell activation; however, our knowledge of viral hindrance of B cell responses of important human pathogens is lacking. Here, we define mechanisms whereby chikungunya virus (CHIKV), a mosquito-transmitted RNA virus that causes outbreaks of acute and chronic arthritis in humans, hinders dLN antiviral B cell responses. Infection of WT mice with pathogenic, but not acutely cleared CHIKV, induced MyD88-dependent recruitment of monocytes and neutrophils to the dLN. Blocking this influx improved lymphocyte accumulation, dLN organization, and CHIKV-specific B cell responses. Both inducible nitric oxide synthase (iNOS) and the phagocyte NADPH oxidase (Nox2) contributed to impaired dLN organization and function. Infiltrating monocytes expressed iNOS through a local IRF5- and IFNAR1-dependent pathway that was partially TLR7-dependent. Together, our data suggest that pathogenic CHIKV triggers the influx and activation of monocytes and neutrophils in the dLN that impairs virus-specific B cell responses.

Introduction

Draining lymphoid organs have a critical role in the initiation of effective adaptive immune responses to vaccines and pathogens. B cells and virus-specific antibodies (Abs) are important arms of the adaptive immune response for limiting viral dissemination and controlling viral infections. The antiviral Ab response is initiated in the draining lymph node (dLN) through a series of molecular interactions and cellular movements that require intact lymphoid tissue architecture and function. Naïve B cells enter the dLN through high endothelial venules (HEVs) and then migrate to defined follicles subjacent to the dLN subcapsular sinus (SCS) [1]. B cells acquire viral antigen through interactions with SCS macrophages which readily acquire viral antigens borne by the afferent lymphatics [2]. After activation, B cells migrate to the borders of the B cell follicle and to the interfollicular region of the dLN to encounter CD4+ T cells. Some of these activated, proliferating B cells go on to form plasmablasts, while others persist in tight clusters in the follicle, forming germinal centers (GCs) [3, 4].

Some viruses have evolved strategies to subvert humoral immune responses in the dLN. Lymphocytic choriomeningitis virus (LCMV), a prototypical mouse pathogen, impairs Ab responses in the dLN via a number of mechanisms, including the recruitment of inflammatory monocytes that promote B cell apoptosis [5]. Vaccinia virus disrupts the dLN SCS macrophage layer, preventing antigen acquisition and subsequent B cell responses [6]. In humans, HIV infection of CD4+ T cells results in loss of follicular T helper cell responses and impaired Ab production [7].

Arthritogenic alphaviruses, including chikungunya virus (CHIKV), Mayaro virus, and Ross River virus, are emerging public health concerns. CHIKV, in particular, re-emerged in 2004 to cause massive outbreaks of disease affecting millions [8]. Acute CHIKV disease presents with rapid onset of fever and rash, with severe joint swelling and arthralgia. A high proportion of individuals develop chronic arthralgia or arthritis that lasts for months to years after initial infection [9]. Following CHIKV infection, the humoral response reduces acute disease severity and controls viremia [10], and passive transfer of immune serum or monoclonal antibodies protects from fatal CHIKV disease [11-13]. However, B cells and Ab are unable to mediate complete clearance of CHIKV from all sites of infection [14-22]. Thus, chronic CHIKV disease in musculoskeletal tissue may be driven by failure of the adaptive immune response to prevent or control viral persistence.

Clearance of the attenuated CHIKV strain 181/25 from musculoskeletal tissue depends on virus-specific Ab production. In comparison, pathogenic CHIKV strains persist in joint-associated tissue of immunocompetent mice [14, 15], suggesting that the development of virus-specific B cell responses may be impaired during CHIKV infection. Indeed, we previously found that pathogenic CHIKV strains disable immune responses in the dLN of infected mice by impairing naïve lymphocyte entry and expansion of HEVs [23]. Additionally, the dLN exhibited follicular disorganization, with a loss of the B-T cell border and poor GC formation following pathogenic CHIKV infection. However, the mechanisms leading to architectural disruption and impaired lymphocyte accumulation remained to be defined.

In this study, we find that a MyD88-dependent influx of monocytes and neutrophils to the dLN disrupts structural organization, impairs lymphocyte accumulation, and diminishes downstream antiviral B cell responses to pathogenic CHIKV infection in a manner partially dependent on expression of inducible nitric oxide synthase (iNOS) and the phagocyte NADPH oxidase (Nox2). We also identify a role for local IRF5-driven type I IFN signaling in the dLN that is triggered by TLR7 and one or more additional TLR(s) and promotes iNOS expression in infiltrating monocytes. Overall, our data suggest that the rapid, early influx of monocytes and neutrophils detrimentally impacts the B cell response to pathogenic CHIKV infection through a novel IRF5-dependent mechanism.

Results

Pathogenic CHIKV infection triggers recruitment of monocytes and neutrophils to the dLN

In previous studies, we found that infection of mice with the attenuated and acutely cleared CHIKV strain 181/25 [14, 24] resulted in dLN hypertrophy, accumulation of lymphocytes, and formation of GCs [23], all hallmarks of appropriate innate and adaptive immune responses to the infection. In contrast, the dLN of mice infected with pathogenic, persistent CHIKV strains became highly disorganized as early as 3 days post-infection (dpi), and failed to accumulate lymphocytes or develop GCs due in part to disruption of HEV-mediated lymphocyte recruitment [23]. Consistent with the robust response of the dLN to attenuated 181/25 infection, the presence of LNs is essential for clearance of 181/25 infection from joint-associated tissue (). To elucidate the mechanisms that lead to the rapid disorganization and function of the dLN following pathogenic CHIKV infection, we characterized cell populations at early times post-infection with pathogenic (parental strain AF15561) or attenuated (derivative strain 181/25) CHIKV, which differ by only five amino acids across the genome [25]. Pathogenic, but not attenuated CHIKV infection, resulted in increased numbers of monocytes (CD11b+Ly6ChiLy6G-) and neutrophils (CD11bhiLy6C+Ly6G+) in the blood and dLN within 24 h of infection (). The influx of monocytes and neutrophils into the dLN preceded their arrival in the foot, the site of virus inoculation (), which occurs between 3 and 5 dpi [26]. In contrast, few monocytes and neutrophils were present in the dLNs of mock-infected mice or mice inoculated with the attenuated CHIKV strain (). Consistent with our flow cytometric analysis, immunofluorescence staining of dLN sections at 1 dpi detected a large population of Gr-1+ monocytes and neutrophils in the dLNs of mice infected with pathogenic CHIKV. These cells were mostly localized within the SCS and medullary sinuses, with a smaller population of monocytes and neutrophils within the B cell follicles (). Thus, during pathogenic, but not attenuated, CHIKV infection, a rapid influx of monocytes and neutrophils into the dLN precedes its extensive disorganization.

Myeloid cells infiltrate the dLN early after pathogenic CHIKV infection.

C57BL/6 mice were inoculated with PBS (mock) or 103 plaque forming units (PFU) of CHIKV 181/25 or AF15561 in the left footpad, and the dLN was analyzed at 24 hpi. (A) Representative flow cytometry plots and numbers of (B) CD11b+Ly6Chi monocytes or (C) CD11bhiLy6C+Ly6G+ neutrophils in the dLN. (D) Frozen dLN sections were stained for ERTR7+ stromal cells (red), B220+ B cells (blue), or Gr-1+ monocytes and neutrophils (white). Errors bars represent mean ± SEM. Data are combined from or representative of 5 (A-C) or 2 (D) independent experiments (n = 15–21 (A-C) or 2–3 (D) mice per group). Statistical significance was determined by one-way ANOVA with Tukey’s post-test (*, P < 0.05; ***, P < 0.001).

Monocyte and neutrophil influx causes reduced lymphocyte accumulation and dLN disorganization

Pathogenic CHIKV infection results in decreased accumulation of naïve lymphocytes and extensive lymphocyte relocalization in the dLN [23]. Because monocyte and neutrophil infiltration of the dLN preceded the disruption of lymphocyte populations ( and [23]), we hypothesized that accumulation of myeloid cells in the dLN might disrupt its architecture. To prevent the early influx of monocytes and neutrophils into the dLN, we treated mice with a single dose of anti-Gr-1 monoclonal antibody (mAb) the day before inoculation with pathogenic CHIKV, which effectively depleted monocytes and neutrophils from the circulation () and the dLN (). Remarkably, a single anti-Gr-1 mAb treatment prior to pathogenic CHIKV infection restored total cell numbers at 5 dpi in the dLN to levels nearly equivalent to those detected during acutely cleared CHIKV infection (), an effect that was due principally to changes in B and CD4+ T cell numbers (). CD8+ T cell numbers in anti-Gr-1-treated mice remained lower than in mice infected with the attenuated CHIKV strain (). The failure to fully restore CD8+ T cell numbers may reflect some effect of the anti-Gr-1 mAb on CD8+ T cells, some of which express Gr-1 upon activation [27]. Depletion of monocytes and neutrophils prior to attenuated CHIKV infection did not affect total cell numbers in the dLN at 5 dpi ().

Delaying the early influx of myeloid cells to the dLN prevents lymphocyte depletion and restores dLN architecture.

C57BL/6 mice were left untreated or treated with 300–500 μg of IgG2b isotype control mAb or anti-Gr-1 mAb via intraperitoneal injection one day prior to inoculation with 103 PFU of CHIKV 181/25 or AF15561 in the left footpad. (A) Representative flow cytometry plots and percentages of (B) CD11b+Ly6Chi monocytes or (C) CD11bhiLy6C+Ly6G+ neutrophils in the blood. (D) Representative flow cytometry plots and numbers of (E) CD11b+Ly6Chi monocytes or (F) CD11bhiLy6C+Ly6G+ neutrophils in the dLN. Data are combined from two independent experiments (n = 3 (mock) to 7 per group). At 5 dpi, the dLN was analyzed for (G) total cells, (H) CD19+B220+ B cells, (I) TCRβ+CD4+ T cells, and (J) TCRβ+CD8+ T cells. Data are combined from 3 independent experiments (n = 4 (mock) or 11 per group). (K) Frozen dLN sections were stained for ERTR7+ stromal cells (red), B220+ B cells (blue), or CD8+ T cells (green). Errors bars represent mean ± SEM. Data in (G-J) are combined from 3 independent experiments. Data in (K) are representative of 2 independent experiments with 4–5 LNs per group. Statistical significance was determined by one-way ANOVA with Tukey’s post-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Pathogenic CHIKV infection causes marked disorganization of lymphocyte populations within the dLN [23], with paracortical relocalization of B cells, diffuse positioning of CD8+ T cells, and loss of a well-defined B-T cell border by 5 dpi. In mice treated with anti-Gr-1 mAb prior to pathogenic CHIKV infection, we observed improved follicular organization and a defined B-T cell border compared with the isotype control group (). Together, these data suggest that the early influx of monocytes and neutrophils into the dLN following pathogenic CHIKV infection results in both impaired lymphocyte accumulation and altered lymphocyte localization.

To evaluate which specific Gr-1+ cell type, monocytes or neutrophils, was responsible for the defect in lymphocyte accumulation in the dLN following pathogenic CHIKV infection, we treated mice with an anti-Ly6G mAb to deplete neutrophils alone or an anti-CCR2 mAb [28] to deplete Ly6Chi monocytes alone. In each case, either neutrophils () or monocytes () were specifically depleted from the circulation and the dLN. As we did not observe restoration of dLN total cell numbers at 5 dpi in mice depleted of either neutrophils () or Ly6Chi monocytes (), the influx of either cell type likely can prevent lymphocyte accumulation in the dLN.

Monocytes or neutrophils prevent lymphocyte accumulation in the dLN during pathogenic CHIKV infection.

(A-F) C57BL/6 mice were treated with 300 μg of IgG2a isotype control Ab or anti-Ly6G mAb via intraperitoneal injection one day prior to inoculation with 103 PFU of CHIKV AF15561 in the left footpad. Representative flow cytometry plots of CD11b+Ly6Chi monocytes or CD11bhiLy6C+Ly6G+ neutrophils in the (A) blood or (B) dLN. At 5 dpi, the dLN was analyzed for (C) total cells, (D) CD19+B220+ B cells, (E) TCRβ+CD4+ T cells, and (F) TCRβ+CD8+ T cells. (G-L) C57BL/6 mice were treated with 50 μg of IgG2b isotype control mAb or anti-CCR2 mAb via intraperitoneal injection one day prior to inoculation with 103 PFU of CHIKV AF15561 in the left footpad. Representative flow cytometry plots of CD11b+Ly6Chi monocytes or CD11bhiLy6C+Ly6G+ neutrophils in the (G) blood or (H) dLN. At 5 dpi, the dLN was analyzed for (I) total cells, (J) CD19+B220+ B cells, (K) TCRβ+CD4+ T cells, and (L) TCRβ+CD8+ T cells. Errors bars represent mean ± SEM. Data are combined from two independent experiments (n = 6–9 per group). Statistical significance was determined by Student’s t-test (n.s., not significant).

Early monocyte and neutrophil influx into the dLN inhibits subsequent GC formation and neutralizing Ab responses

In comparison with attenuated CHIKV infection, pathogenic CHIKV infection induces poor GC formation in the dLN and a diminished neutralizing serum Ab response [14, 23]. We hypothesized that the enhanced lymphocyte accumulation and organization in the dLN of mice depleted of monocytes and neutrophils prior to pathogenic infection would improve GC formation. To investigate this idea, mice were treated with a single dose of anti-Gr-1 or isotype control mAb one day prior to pathogenic CHIKV infection, and GC responses in the dLN were evaluated at 14 dpi by staining sections with the GC B cell marker GL7. Anti-Gr-1 mAb-treated mice had increased numbers of GCs per dLN, and the GCs were larger than those of the isotype control group (), resulting in an increased total GC area in the dLN (). Flow cytometry analysis also showed an increased percentage and number of CD95+GL7+ GC B cells in the dLN of mice infected with pathogenic CHIKV and treated with anti-Gr-1 mAb (). Early depletion of monocytes and neutrophils also resulted in greater numbers of CD138+IgDlo plasma cells (PCs) () and increased the number of CHIKV-specific Ab-secreting cells (ASCs) in the dLN (). The effects of anti-Gr-1 mAb treatment were most pronounced in the dLN, as we observed modest effects on the percentages of GC B cells and plasma cells in the left inguinal LN and the spleen (). Anti-Gr-1 mAb treatment had no effect on the amount of total CHIKV-specific IgG present in serum at day 7, 14, and 28 dpi (). However, consistent with the effects of anti-Gr-1 mAb treatment on GC formation, we observed an increase in CHIKV neutralization activity in the serum of anti-Gr-1-treated mice compared with the isotype control group at 28 dpi (). Together, these data demonstrate that early depletion of monocytes and neutrophils during pathogenic CHIKV infection improves the endogenous, polyclonal virus-specific GC B cell responses in the dLN and, despite predominantly impacting the dLN, leads to a modest but detectable improvement in serum Ab neutralization activity.

Depletion of myeloid cells improves dLN B cell responses.

C57BL/6 mice were treated with 300–500 μg IgG2b isotype control mAb or anti-Gr-1 mAb via intraperitoneal injection one day prior to inoculation with 103 PFU of CHIKV AF15561 in the left footpad. (A) At 14 dpi, dLN sections were stained for ERTR7+ stromal cells, B220+ B cells, and GL7+ GC B cells. (B) Area per GC, (C) Number of GCs per LN section, and (D) total GC area per LN were determined. (E) Representative flow cytometry plots of GL7+CD95+ GC B cells (gated on CD19+B220+ cells), (F) percentage and (G) total number of GC B cells in the dLN at 14 dpi. (H) Representative flow cytometry plots of CD138+IgD- plasma cells (gated on CD19+), (I) percentage and (J) total number of plasma cells in the dLN at 14 dpi. (K) Number of CHIKV-specific IgG+ antibody secreting cells (ASCs) in the dLN at 14 dpi. Serum collected at the indicated timepoints was assayed for (L) total CHIKV-specific IgG by ELISA (expressed as fold change in IgG endpoint over the IgG2b group) and (M) neutralizing activity of CHIKV by focus reduction neutralization test (FRNT, expressed as fold change in FRNT50 over the IgG2b group). Errors bars represent mean ± SEM. Data in (A-D) are derived from 5–6 LNs per group with 7–12 LN sections analyzed per group. Data in (E-M) are derived from 2–3 independent experiments with 7–10 mice per group. Statistical significance was determined by Student’s t-test in B-D (note that significance is only displayed for comparison of IgG2b and α-Gr-1 groups), F-G, I-J, and K; or by two-way ANOVA with Bonferroni’s post-test in L-M (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Mechanisms of pathogenic CHIKV-induced monocyte and neutrophil recruitment

We next investigated the molecular pathways by which monocytes and neutrophils are recruited into the dLN following pathogenic CHIKV infection. Recruitment of Ly6Chi monocytes to inflamed tissue or the dLN in response to viral infection is reportedly CCR2- and type I IFN-dependent [5, 29]. Accordingly, we first investigated the role of type I IFN signaling in the accumulation of monocytes and neutrophils in the dLN following pathogenic CHIKV infection. As shown in , the total number of dLN cells following pathogenic CHIKV infection was reduced in Ifnar1 compared with wild-type (WT) mice; type I IFN signaling normally inhibits lymphocyte egress [30], but this does not occur in mice lacking the type I IFN receptor and likely contributes to the reduced cell numbers. The percentage of neutrophils was increased in the dLN of Ifnar1 mice compared with WT mice after pathogenic CHIKV infection (), which resulted in a similar total number of neutrophils in the dLN of WT and Ifnar1 mice () despite the overall decrease in total number of dLN cells. The frequencies of Ly6Chi monocytes were similar in the dLN of CHIKV-infected WT and Ifnar1 mice () although the total number of monocytes in the dLN was reduced in Ifnar1 mice.

Mechanisms of pathogenic CHIKV-induced myeloid cell recruitment to the dLN.

Mice were inoculated with 103 PFU of CHIKV AF15561 in the left footpad. At 24 hpi, total cells, CD11bhiLy6C+Ly6G+ neutrophils, and CD11b+Ly6Chi monocytes in the dLN were enumerated in (A-F) WT or Ifnar1 mice, (G-L) WT or Myd88 mice, or (M-R) WT mice treated with 200 μg IgG or α-IL-1R Ab at the time of infection. Errors bars represent mean ± SEM. Data are combined from 2–4 independent experiments (n = 7–16 mice per group). Statistical significance was determined by Student’s t-test (*, P < 0.05; ***, P < 0.001).

Because TLR ligands can promote monocyte egress from the bone marrow to sites of infection [31], we evaluated neutrophil and monocyte recruitment to the dLN of mice deficient in MyD88, the canonical adaptor for many TLRs and the interleukin-1 receptor (IL-1R) family [32]. Genetic deletion of MyD88 had minimal impact on the total number of cells in the dLN at 24 hpi (). However, MyD88-dependent signals were required for and contributed to neutrophil () and monocyte () recruitment, respectively, to the dLN following pathogenic CHIKV infection. Mice treated with anti-IL-1R blocking mAb at the time of infection showed no change in total cells in the dLN compared with control IgG-treated mice (), but had reduced neutrophils (percentage and total number; ), indicating that the MyD88-IL-1R signaling axis regulates the rapid accumulation of neutrophils in the dLN during pathogenic CHIKV infection. In contrast, the percentage and total number of monocytes were unchanged in the anti-IL-1R-treated group compared with the IgG-treated group (). Together, these data demonstrate that in response to pathogenic CHIKV infection 1) type I IFN signaling inhibits and IL-1R signaling promotes, neutrophil recruitment to the dLN, and 2) IL-1R-independent, MyD88-dependent signal(s) promote the recruitment of monocytes to the dLN.

Nos2 and Nox2 contribute to reduced lymphocyte numbers and disorganization in the dLN

Myeloid cells can suppress immune responses by a variety of mechanisms, including through the production of nitric oxide (NO) via the action of inducible nitric oxide synthase (iNOS, encoded by Nos2) [33]. Moreover, superoxide derived from the activity of the phagocyte NADPH oxidase (NOX2, encoded by gp91phox, also known as Cybb) can combine with NO to form the reactive nitrogen species peroxynitrite (ONOO-), which also can suppress immune responses [34, 35]. Based on this knowledge, we hypothesized that monocytes and neutrophils infiltrating the dLN disrupt lymphocyte accumulation and tissue organization through the actions of iNOS and NOX2. Consistent with the lack of effect of monocyte and neutrophil depletion on accumulation of lymphocytes in the dLN following attenuated CHIKV infection (), total cell numbers in the dLN were unchanged in Nos2 and Nox2 mice infected with attenuated CHIKV (). However, total cell numbers in the dLN were partially restored in Nos2 and Nox2 mice inoculated with pathogenic CHIKV compared with WT mice (), suggesting that NO and superoxide contribute to reduced lymphocyte numbers in the dLN during pathogenic CHIKV infection. In addition to partial restoration of lymphocyte numbers, the dLNs of Nos2 and Nox2 mice infected with pathogenic CHIKV showed improved follicular and paracortical organization, and a more clearly defined B-T cell border compared with the dLN of WT mice (). These data suggest that both Nos2 and Nox2 contribute to the disruption of lymphocyte organization that occurs during pathogenic CHIKV infection.

Nos2 and Nox2 contribute to disrupted dLN architecture and lymphocyte depletion.

WT, Nos2, or Nox2 (gp91phox) mice were inoculated with PBS or 103 PFU of CHIKV AF15561 in the left footpad. (A) At 5 dpi, total cells in the dLN were enumerated. Data are combined from 3–4 independent experiments (n = 4–16 mice per group). (B) Frozen dLN sections (6–7 LNs per group) were stained for ERTR7+ stromal cells (red), B220+ B cells (blue), or CD8+ T cells (green). (C) At 24 hpi, representative flow cytometry plots and numbers of (D) CD11b+Ly6Chi monocytes or (E) CD11bhiLy6C+Ly6G+ neutrophils in the dLN. (F) Frozen dLN sections were stained for B220+ B cells (blue), or Gr-1+ monocytes and neutrophils (white). (G) Representative flow cytometry plots and (H) numbers of iNOS+ cells among circulating (bottom) or dLN (top) CD11b+Ly6Chi monocytes in WT and Nos2 mice. Errors bars represent mean ± SEM. Data are combined from (A, C-E, G-H) or representative of (B, F) 2–3 independent experiments (n = 4–12 mice per group). Statistical significance in (A–displayed only for comparison between AF15561-infected groups) and (H) was determined by Student’s t-test to compare groups (***, P < 0.001).

The partial restoration of cell numbers and markedly improved lymphocyte organization in the dLN were not due to a difference in early monocyte and neutrophil infiltration or localization at 24 hpi between WT, Nos2, and Nox2 mice (). To define the iNOS-expressing cell type(s) that mediate the Nos2-dependent effects on dLN cell numbers and organization, we analyzed cell populations in the dLN at 24 hpi by flow cytometry. Monocytes elicited by pathogenic CHIKV infection expressed iNOS in the dLN, but not in the blood (). iNOS staining was absent in neutrophils in the dLN (), and was absent in Nos2-/- mice, as expected (). Together, these data indicate that expression of Nos2 and Nox2 contribute to impaired lymphocyte accumulation and dLN disorganization, and that monocytes upregulate iNOS expression following entry into the dLN.

iNOS expression in monocytes is driven by an IFNAR1- and IRF5-dependent pathway

We next evaluated the molecular pathways that promote iNOS expression in monocytes infiltrating the dLN. Many inflammatory stimuli can induce iNOS expression, including type I IFNs [36]. Indeed, type I IFN receptor signaling was required for expression of iNOS in dLN monocytes following CHIKV infection (). Type I IFN signaling and either IRF3 or IRF7 are required for protection from fatal CHIKV infection, with IRF7 responsible for systemic type I IFN production [37, 38]. Unexpectedly, both IRF3 and IRF7 were dispensable for iNOS induction in dLN monocytes (). Instead, Irf3Irf5Irf7 triple knockout mice showed diminished monocyte iNOS expression similar to that of Ifnar1 mice, suggesting a requirement for IRF5. The dependency on IRF5 for monocyte iNOS expression was confirmed in Irf5 mice (). Together, these data suggest that IRF5- and IFNAR1-dependent signals act locally in the dLN to induce iNOS expression in infiltrating monocytes during pathogenic CHIKV infection.

Mechanisms of iNOS expression in monocytes recruited to the dLN.

Mice were inoculated with PBS (mock) or 103 PFU of CHIKV AF15561 in the left footpad, and the dLN was analyzed at 24 hpi. (A) Representative flow cytometry plots and (B) percentage of CD11b+Ly6Chi monocytes expressing iNOS in WT, Ifnar1, Irf3Irf7, or Irf3Irf5Irf7 mice. (C) Representative flow cytometry plots and (D) percentage of CD11b+Ly6Chi monocytes expressing iNOS in WT or Irf5 mice. (E) Representative flow cytometry plots and (F) percentage of CD11b+Ly6Chi monocytes expressing iNOS in WT, Tlr7, or Myd88 mice. (G-H) WT mice were treated with 200 μg of IgG or α-IL-1R Ab at the time of infection. (G) Representative flow cytometry plots and (H) percentage of CD11b+Ly6Chi monocytes expressing iNOS. Errors bars represent mean ± SEM. Data are combined from 2–3 independent experiments. Statistical significance is displayed only for comparison between infected groups (mock not included) and was determined by one-way ANOVA with Tukey’s post-test (B, F) or Student’s t-test (D, H) (***, P < 0.001).

iNOS expression in monocytes requires MyD88 and is partially dependent on TLR7

We next assessed the upstream signals required for monocyte expression of iNOS. IRF5 can be activated in a MyD88-dependent manner by TLR7 or TLR9 agonists [39-41]. In the context of some inflammatory stimuli, TLR3-mediated TRIF signaling can synergize with MyD88 for full IRF5 activation [42, 43]. However, pathogenic CHIKV-induced iNOS expression in dLN monocytes was independent of TRIF (). Instead, iNOS expression in infiltrating monocytes was completely dependent on MyD88 (), and partially dependent on TLR7 (), suggesting that multiple MyD88-dependent signals contribute to IRF5 activation. To determine whether the other MyD88-dependent signal responsible for iNOS induction originated from IL-1R, we treated mice with an IL-1R blocking mAb at the time of infection. IL-1R blockade did not alter the percentage of iNOS-expressing monocytes in the dLN (). Together, these data suggest that TLR7 and one or more additional MyD88-dependent TLRs activate IRF5 to drive iNOS expression in dLN infiltrating monocytes following pathogenic CHIKV infection.

An IRF5-dependent pathway is activated locally in the dLN

The upregulation of iNOS in monocytes in the dLN, but not in circulation, and the requirement for IFNAR1 and IRF5 in this induction suggested the presence of an IRF5-dependent type I IFN pathway acting locally in the dLN. To investigate this idea, we infected WT, Irf3Irf7, and Irf3Irf5Irf7 mice with pathogenic CHIKV. At 24 hpi, we measured the induction of interferon stimulated genes (ISGs) including Ccl2, Cxcl10, Ifng, Il6, Ifit1, and Rsad2 mRNA [44, 45] in the inoculated foot and in the dLN by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Each of these ISGs was upregulated in both the foot and dLN of CHIKV-infected WT mice at this timepoint (). However, upregulation was differentially dependent on IRF3/IRF7 or IRF5 in each tissue. In the inoculated foot, upregulation of all ISGs analyzed was dependent on IRF3/IRF7 (). However, in the dLN, the induction of Ccl2, Cxc10, Ifng, and Il6 mRNA was similar between WT and Irf3Irf7 mice (), whereas Ifit1 and Rsad2 upregulation in the dLN were only partially dependent on the presence of IRF3/IRF7 (). Instead, for each ISG, the combined deficiency of IRF3, IRF5, and IRF7 resulted in reduced upregulation in the dLN compared with WT or Irf3Irf7 mice (). Together, these data demonstrate that induction of multiple ISGs at the site of inoculation is dependent on IRF3/IRF7, whereas an IRF5-dependent ISG induction pathway is activated in the dLN.

An IRF5-dependent pathway is activated locally in the dLN.

WT, Irf3Irf7, or Irf3Irf5Irf7 mice were inoculated with PBS (mock) or 103 PFU of CHIKV AF15561 in the left footpad. At 24 hpi, the inoculated foot and the dLN were collected. qRT-PCR was used to analyze mRNA expression of (A) Ccl2, (B) Cxcl10, (C) Ifng, (D) Il6, (E) Ifit1, and (F) Rsad2. Error bars represent mean ± SEM. Data (8–9 mice per group) are combined from 2 independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01, ***P < 0.001.

Discussion

Our results demonstrate that a rapid influx of Ly6Chi monocytes and neutrophils into the dLN triggered by infection with pathogenic, persistent strains of CHIKV reduces the accumulation of lymphocytes, disrupts lymphocyte organization, and impairs the development of virus-specific B cell responses. Preventing the early influx of Ly6Chi monocytes and neutrophils into the dLN improved accumulation of lymphocytes and follicular organization and enhanced dLN GC formation, plasma cell differentiation, and CHIKV-specific serum neutralizing antibody responses. Lymphocyte accumulation and dLN organization was improved in CHIKV-infected mice lacking Nos2 (iNOS) or the phagocyte NADPH oxidase Nox2. Monocytes, but not other cell types, upregulated iNOS following entry into the dLN in a manner dependent on IFNAR1, IRF5, and MyD88, and partially dependent on TLR7. Although IRF3/IRF7-dependent type I IFN production is required for protection from fatal CHIKV infection [37, 46], we identified an IRF5-dependent pathway acting locally within the dLN following pathogenic CHIKV infection that drives activation of infiltrating monocytes.

Following infection or vaccination, innate immune cells are recruited to lymphoid tissues [5, 47–49]. Myeloid-derived cells can have suppressive effects on adaptive immunity, depending on the local inflammatory milieu. For example, monocytes recruited to the dLN of LCMV-infected mice co-localize with and deplete virus-specific B cells in the interfollicular area [5]. Here, we show that pathogenic, but not acutely cleared CHIKV, similarly induces rapid recruitment of monocytes and neutrophils to the dLN. During pathogenic CHIKV infection, recruited monocytes and neutrophils localized to the SCS and medullary sinuses, with some cells observed within B cell follicles. Ongoing studies are aimed at determining the cell types in the dLN with which monocytes and neutrophils interact, and how their localization determines the subsequent disruption of dLN architecture following infection.

We previously observed that pathogenic CHIKV infection blunts the accumulation of lymphocytes in the dLN by impairing recruitment of naïve lymphocytes from the circulation [23]. Failure to recruit new lymphocytes was due to reduced function of high endothelial venules (HEVs) and decreased production of homeostatic chemokines. Preventing the early influx of monocytes and neutrophils into the dLN restored naïve lymphocyte recruitment, as the lymphocyte numbers in the dLN were uniformly increased. Moreover, preventing the influx of monocytes and neutrophils into the dLN improved organization of B cell follicles, the T cell area, and the B-T cell border. It remains to be determined whether these infiltrating myeloid cells directly also affect the function of HEVs or the production of homeostatic chemokines. Furthermore, monocyte and neutrophil depletion improved virus-specific B cell responses in the dLN, with more GC B cells and plasma cells generated. This improved GC formation is likely a downstream consequence of boosted lymphocyte numbers and less perturbed dLN organization earlier during infection.

Type I IFNs can promote monocyte recruitment during inflammatory responses [5, 50]. Our data reveal distinct roles for IFNAR1, MyD88, and IL-1R in the recruitment of monocytes and neutrophils to the dLN. Neutrophil recruitment was inhibited by IFNAR1 signaling, but required IL-1R-MyD88 signaling. These findings agree with prior reports demonstrating IL-1R-dependent neutrophil recruitment in response to several inflammatory stimuli [51-53], and a single report showing IFNAR1-mediated inhibition of neutrophil recruitment to inflamed lungs following influenza A virus infection [54]. In contrast to neutrophils, monocyte recruitment to the dLN following pathogenic CHIKV infection was independent of IFNAR1 and IL-1R signaling. However, the percentage and number of monocytes in the dLN was reduced in MyD88-deficient mice, indicating that one or more TLRs likely drive monocyte recruitment.

Total lymphocyte numbers in the dLN were increased in the dLN of Nos2 and Nox2 mice compared with WT mice following pathogenic CHIKV infection. In addition, and in contrast to WT mice, pathogenic CHIKV infection had minimal effects on the organization of B cell follicles and the T cell zone in the dLN of Nos2 and Nox2 mice. Flow cytometric analysis revealed that monocytes, but not neutrophils, upregulated iNOS expression following entry into the dLN. During LCMV infection of mice, monocytes infiltrating the dLN induced apoptosis in virus-specific and bystander B cells in a Nos2-dependent manner [5]. Studies with LCMV also observed B cell depletion in lymphoid tissue, however, individual roles for Nos2 [55] or Gr-1+ cells [56] were not defined. In contrast to LCMV infection, extensive lymphocyte death or an increase in apoptotic lymphocytes does not occur in the dLN following pathogenic CHIKV infection [23], suggesting that the infiltrating monocytes do not impair B cell responses by inducing death of lymphocytes. Additionally, lymphocyte numbers were restored only partially in mice deficient for Nos2 or Nox2, suggesting that the combined action of Nos2 and Nox2 or other monocyte and/or neutrophil-derived factors contribute to the impairment of naive lymphocyte accumulation and disruption of LN organization. Depletion of either neutrophils or monocytes alone did not restore total lymphocyte numbers in the dLN, indicating that influx of either cell type is sufficient to prevent accumulation of naïve lymphocytes. Although the improved dLN organization and lymphocyte accumulation observed in Nos2-deficient mice could be ascribed to lack of iNOS expression uniquely in monocytes, the mechanism(s) by which neutrophils impair lymphocyte accumulation and disrupt dLN architecture remain to be determined.

Type I IFN signaling can modulate monocyte activation and differentiation, with context-dependent pro-inflammatory or anti-inflammatory effects [36, 54, 57, 58]. Although not required for recruitment of monocytes to the dLN after pathogenic CHIKV infection, type I IFN signaling drives iNOS expression in monocytes following entry into the dLN. Furthermore, IRF5, but not IRF3 or IRF7, was responsible for induction of iNOS. IRF3 or IRF7 are required for protection from fatal CHIKV infection, with systemic type I IFN production being driven by the IRF7 response [37, 38]. However, the contribution of type I IFN signaling and additional IRFs in other aspects of alphavirus pathogenesis remains poorly defined. Our work suggests that an IRF5-dependent pathway acts locally in draining lymphoid tissue following infection with CHIKV, whereas IRF3/7-dependent pathways are activated at the site of inoculation. Studies in IRF5-overexpressing cells found that IRF5 induces a distinct subset of IFNα genes compared with other IRFs, such as IRF7 [59]. In addition to IFNα, IRF5 also promotes the transcription of genes encoding proinflammatory cytokines, such as Il12b, Tnfa, and Il6 [41] and several chemokines [60]. Similar to our observations, IRF5 promotes the induction of several proinflammatory cytokines and chemokines, as well as recruitment and activation of lymphocytes in the dLN following West Nile Virus (WNV) infection of mice [61]. However, these IRF5-dependent responses were ultimately protective by limiting viral spread and promoting optimal B cell immunity. In contrast, our data suggest that pathogenic strains of CHIKV trigger an IRF5-dependent inflammatory pathway in the dLN that activates infiltrating monocytes and neutrophils, ultimately causing disruption of dLN architecture and decreased virus-specific B cell responses.

IRF5 is expressed constitutively in pDCs, classical or conventional DCs (cDCs), M1 macrophages, and B cells, with further upregulation following activation [41, 59, 62–65]. Upon activation with TLR ligands, IRF5 expression also can be induced in human monocytes, neutrophils, and macrophages [41, 62, 66]. Thus, many immune cell types can express IRF5, either constitutively or upon activation. Viral infections or agonists of TLR7 or TLR9 can activate IRF5 in DCs in vitro in a MyD88-dependent manner [39-41]. Following pathogenic CHIKV infection, we observed a partial role for TLR7 in iNOS upregulation, suggesting that recognition of endosomal viral RNA contributes to monocyte activation. However, our data suggest role(s) for other MyD88-dependent TLR(s) in activating IRF5 to promote iNOS expression in infiltrating monocytes. One or more of these TLRs may be activated by endogenous damage-associated molecular patterns (DAMPs) released from damaged or dying cells during the early stages of CHIKV infection, either at the site of inoculation or in the dLN. The cell-intrinsic requirements for TLR(s), IRF5, and IFNAR1 expression in dLN monocyte iNOS upregulation during CHIKV infection remain to be determined through the use of conditional knockout mice.

In summary, we found that a rapid influx of Ly6Chi monocytes and neutrophils to the dLN impairs lymphocyte accumulation, dLN organization, and downstream antiviral B cell responses to pathogenic CHIKV infection in a manner partially dependent on Nos2 and Nox2. Furthermore, we identified a role for MyD88-dependent IRF5-driven type I IFN signaling in the dLN that promotes iNOS expression in infiltrating monocytes. Our findings reveal an additional mechanism that dampens the development of the anti-CHIKV Ab response during pathogenic CHIKV infection, and underscore how local innate immune responses in draining lymphoid tissue dictate the magnitude of downstream adaptive immunity.

Materials and methods

Ethics

This study was conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and the AVMA Guidelines for the Euthanasia of Animals. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University School of Medicine (Assurance Number: A3269-01) or at the Washington University School of Medicine (Assurance Number A3381-01). Experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by thoracotomy.

Mouse experiments

WT C57BL/6 (000664), Nos2 (002609; B6.129P2-Nos2tm1Lau), gp91 (002365; B6.129S6-Cybbtm1Din), Tlr7 (008380; B6.129S1-Tlr7), Myd88 (009088; B6.129P2(SJL)-Myd88), and LT (002258; B6.129S2-Lta/J) mice were obtained from The Jackson Laboratory. Irf5 mice were a gift of T. Taniguchi (Tokyo, Japan), obtained from I. Rifkin (Boston, MA), and were backcrossed for eight generations. After detection of a homozygous Dock2 mutation in this line, it was backcrossed for five additional generations to obtain Irf5 Dock2 mice [61]. Irf3Irf5Irf7 mice were a gift from Dr. Sujan Shresta (La Jolla Institute for Immunology) and originally generated at Washington University [45]. Nos2, gp91 (Nox2), Tlr7, Myd88, Irf5, Irf3Irf7, Irf3Irf5Irf7, and Ifnar1 mice were bred and housed at the University of Colorado School of Medicine or Washington University under specific pathogen-free conditions. Mice were used at 3–4 weeks of age. Virus inoculations were performed under isoflurane anaesthesia and all efforts were made to minimize animal suffering. All mouse experiments were performed in an animal biosafety level 3 laboratory.

Mice were anesthetized with isoflurane and inoculated with 103 PFU of CHIKV subcutaneously in the left rear footpad in a volume of 10 μL of PBS/1% FBS. Mice were sacrificed after exposure to isoflurane vapors followed by thoracotomy and perfusion with PBS at the indicated time points. In some experiments, mice were treated with 300–500 μg of IgG2b isotype control Ab (LTF-2), anti-Gr-1 mAb (RB6-8C5), IgG2a isotype control Ab (2A3), or anti-Ly-6G mAb (1A8) (all from BioXCell) i.p. the day prior to infection. For IL-1R blockade, mice were treated with 200 μg Armenian hamster IgG control Ab or anti-IL-1R Ab (JAMA-147) (both from BioXCell) at the time of infection.

Cell lines and viruses

BHK-21 cells (American Type Culture Collection) were cultured at 37°C in Minimum Essential Medium Alpha (MEMα) supplemented with 10% fetal bovine serum (FBS) and 10% tryptose phosphate broth (TPB). Vero cells (American Type Culture Collection) were cultured at 37°C in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) with 10% FBS.

CHIKV cDNA clones encoding 181/25 and AF15561 have been described previously [67]. Stocks of infectious CHIKV strains were generated from cDNA clones and titered by plaque assay on BHK-21 cells as previously described [68]. The number of particles/mL of each virus stock was determined by qRT-PCR. All experiments were performed under biosafety level 3 conditions.

Isolation of cells from lymphoid tissue and flow cytometry

The left popliteal LN was gently homogenized in a Biomasher II tissue homogenizer (Kimble-Chase) in RPMI 1640 (HyClone) supplemented with 10% FBS. For flow cytometric analysis of infiltrating footpad or dLN cells, 2.5 mg/ml collagenase type I (Worthington Biochemical) and 17 μg/ml DNase I (Roche) were added to RPMI 1640 with 10% FBS, and samples were incubated for 1 h at 37°C with shaking (130 rpm). Following erythrocyte lysis (footpad only), cells were passed through a 100 μm cell strainer (BD Falcon) and total viable cells were determined by trypan blue exclusion. For analysis of circulating immune cells, blood was collected from the inferior vena cava into 50 μL heparin. Following erythrocyte lysis, cells were stained as below.

Single cell suspensions were incubated with anti-mouse FcγRIII/II (2.4G2; BD Pharmingen) for 20 min on ice and then stained with antibodies against the following cell surface markers in 1X PBS/2% FBS for 1 h on ice: CD45 (30-F11), CD11b (M1/70), Ly-6C (HK1.4), Ly-6G (1A8), CD19 (6D5), B220 (RA3-6B2), TCRβ (H57-597), CD4 (RM4-5), CD8 (53–6.7), CD95 (Fas; 15A7), GL7 (GL7), IgD (11-26c.2a), and CD138 (281–2) (all Abs from BioLegend, BD Bioscience, or Thermo Fisher). Cells were washed three times in PBS/2% FBS and fixed overnight in 1X PBS/1% PFA prior to analysis. For intracellular analysis of iNOS expression, cells were stained first with antibodies against cell surface markers, fixed for 15 min at RT in PBS/1% paraformaldehyde (PFA)/0.1% saponin, and then stained with anti-iNOS Ab (clone CXNFT; Thermo Fisher) in PBS/2% FBS/0.1% saponin for 1 h at 4°C. Cells were washed three times in PBS/2% FBS/0.1% saponin, fixed overnight in 1X PBS/1% PFA, and analyzed on a BD LSR Fortessa cytometer using FACSDiva software. Further analysis was performed using FlowJo software (Tree Star).

CHIKV ELISA and focus reduction neutralization test (FRNT)

CHIKV-specific antibodies in mouse sera were measured using a virion-based ELISA as described [14]. Endpoint titers were defined as the reciprocal of the last dilution to have an absorbance two times greater than background. Blank wells receiving no serum were used to quantify background signal. Fold change was calculated by dividing the IgG endpoint of each sample by the average IgG endpoint of the group receiving IgG2b control mAb.

For FRNT assays, Vero cells were seeded in 96-well plates. Serum samples were heat-inactivated and serially diluted in DMEM/F12 medium with 2% FBS in 96-well plates. Approximately 100 focus-forming units (FFU) of virus stock was added to each well and the serum plus virus mixture was incubated for 1 h at 37°C. At the end of 1 h, medium was removed from Vero cells and serum sample + virus mixture was added for 2 h at 37°C. After 2 h, sample was removed and cells were overlaid with 0.5% methylcellulose in MEM/5% FBS and incubated 18 h at 37°C. Cells were fixed with 1% PFA and probed with 500 ng/mL of the anti-CHK-11 mAb [12] diluted in 1X PBS/0.1% saponin/0.1% bovine serum albumin (BSA) for 2 h at RT. After washing, cells were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, 1:2000) for 1.5–2 h at RT. After washing, CHIKV-positive foci were visualized with TrueBlue substrate (Fisher) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology). Percent infectivity was calculated compared to a virus only (no serum) control. The FRNT50 value was defined as the reciprocal of the last dilution to exhibit 50% infectivity. Fold change was calculated by dividing the FRNT50 of each sample by the average FRNT50 of the group receiving IgG2b control Ab.

CHIKV IgG enzyme linked immunosorbent assay (ELISPOT)

For quantification of CHIKV-specific IgG+ ASCs, dLNs were removed and processed into a single cell suspension as above. Two-fold dilutions of cells (starting at 1 x 105) were plated on filter plates (EMP Millipore MultiScreen HTS-IP) coated with 108 particles/well of CHIKV AF15561 and incubated for 5 h at 37°C. Biotin-conjugated anti-IgG and streptavidin-conjugated horseradish peroxidase (HRP) (both from Southern Biotech) were used for detection. For development, 200 μL 3-amino-9-ethylcarbazole (AEC) solution (Sigma) was added to 9 ml 0.1 M sodium acetate buffer containing 4 μL 30% H2O2. Spots were analyzed on a C.T.L. ELISPOT Reader using ImmunoSpot 6.1 software (Cellular Technology).

Gene expression analysis from the dLN and foot

The dLN or left foot was dissected and homogenized in TRIzol Reagent (Life Technologies) for RNA analysis with a MagNA Lyser (Roche). RNA was isolated using the PureLink RNA Mini kit (Thermo Fisher) with on-column DNase treatment. Gene expression was quantified by RT-qPCR using available Taqman gene expression assays (Thermo Fisher). Expression of each gene was normalized to 18S and expressed as fold change over mock samples.

Viral RNA analysis

Ankle and spleen tissues were dissected and homogenized in TRIzol Reagent (Life Technologies) for RNA analysis with a MagNA Lyser (Roche). Viral RNA in tissues was quantified by RT-qPCR as previously described [23].

Immunofluorescence and confocal microscopy

Lymph nodes were fixed in 1 mL of phosphate buffer containing 0.1 M L-lysine, 2% PFA, and 2.1 mg/mL NaIO4 at pH 7.4 for 24 h at 4°C, followed by incubation in 30% sucrose phosphate-buffered solution for 48 h, then in 30% sucrose/PBS for 24 hr. LNs were then embedded in optimal-cutting-temperature medium (Electron Microscopy Sciences) and frozen in dry-ice-cooled isopentane. Eighteen-μm sections were cut on a Leica cryostat (Leica Microsystems). Sections were blocked with 5% goat, donkey, bovine, rat or rabbit serum and then stained with one or more of the following: B220 (clone RA3-6B2, ThermoFisher), CD8α (clone 53–6.7, ThermoFisher), ERTR-7 (Rat monoclonal, BioXCell), Gr-1 (clone RB6-8C5, BioXCell), and GL7 (clone GL7, BioLegend). Images were acquired using identical photomultiplier tube (PMT) and laser power settings on a Leica SP5 confocal equipped with HyD detectors (Leica). Confocal microscopy images were performed of the entire popliteal lymph node (representing approximately a 7 mm2 imaged area) and individual fields (tiles) were merged into a single image file. Images were analysed using Imaris v9.02 software (Bitplane). The total number of GCs, area per GC, and total GC area were calculated per 18-μM dLN section automatically using Imaris’ surfaces function based upon GL7 staining. The total GC number was also confirmed manually.

Quantification and statistical analysis

Statistical details for each experiment are found in the corresponding figure legends. Statistical analyses were conducted using GraphPad Prism 6.0.

Peripheral lymph organs are required for clearance of CHIKV 181/25 infection in joint-associated tissue.

WT or congenic lymphotoxin alpha-deficient (LT) C57BL/6 mice were inoculated with 103 PFU of CHIKV 181/25 or AF15561 in the left footpad. At 28 dpi, RNA was extracted from the (A) right ankle and (B) spleen and viral loads were determined by RT-qPCR. Data are from one independent experiment.

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Circulating myeloid cells elicited by pathogenic CHIKV infection infiltrate the dLN before the site of inoculation.

C57BL/6 mice were inoculated with PBS (mock) or with 103 PFU of CHIKV 181/25 or AF15561 in the left footpad. At 24 hpi, the blood and left foot were analyzed by flow cytometry. (A) Representative flow cytometry plots and percentages of (B) CD11b+Ly6Chi monocytes or (C) CD11bhiLy6C+Ly6G+ neutrophils in the blood. (D) Representative flow cytometry plots and numbers of (E) monocytes or (F) neutrophils in the left foot. Data are combined from two (A-C) or five (D-F) independent experiments (n = 4–21 per group). Statistical significance was determined by one-way ANOVA with Tukey’s post-test (***, P < 0.001).

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Anti-Gr-1 mAb treatment does not affect lymphocyte accumulation in dLN following attenuated CHIKV infection.

C57BL/6 mice were treated with 500 μg of IgG2b isotype control mAb or anti-Gr-1 mAb via intraperitoneal injection one day prior to inoculation with 103 PFU of CHIKV 181/25 in the left footpad. At 5 dpi, total cells in the dLN were enumerated. Data are combined from 2 independent experiments.

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The effects of anti-Gr-1 mAb treatment are largely confined to the dLN.

C57BL/6 mice were treated with 300–500 μg IgG2b isotype control mAb or anti-Gr-1 mAb i.p. one day prior to inoculation with 103 PFU of CHIKV AF15561 in the left footpad. (A) Representative flow cytometry plots of GL7+CD95+ GC B cells (gated on CD19+B220+ cells), (B) percentage and (C) total number of GC B cells in the left inguinal LN at 14 dpi. (D) Representative flow cytometry plots of CD138+IgD- plasma cells (gated on CD19+), (E) percentage and (F) total number of plasma cells in the left inguinal LN at 14 dpi. (G) Representative flow cytometry plots of GL7+CD95+ GC B cells (gated on CD19+B220+ cells), (H) percentage and (I) total number of GC B cells in the spleen at 14 dpi. (J) Representative flow cytometry plots of CD138+IgD- plasma cells (gated on CD19+), (K) percentage and (L) total number of plasma cells in the spleen at 14 dpi. Errors bars represent mean ± SEM. Data are derived from 2 independent experiments. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01.

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iNOS expression in monocytes is independent of TRIF.

C57BL/6 mice were inoculated with PBS (mock) or with 103 PFU of CHIKV AF15561 in the left footpad and the dLN was analyzed at 24 hpi. (A) Percentage and (B) representative flow cytometry plots of CD11b+Ly6Chi monocytes expressing iNOS in WT or Trif mice. Data are combined from 2 independent experiments.

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1 Oct 2019

Dear Dr. Morrison,

Thank you very much for submitting your manuscript "MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to CHIKV infection via Irf5, Nos2 and Nox2" (PPATHOGENS-D-19-01494) for review by PLOS Pathogens. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

*customized message from Marco: Hello Thomas, I appologize for the original delay in sending this work out to review, as it was stalled in my feed while I was on vacation. As you will see from the reviews, each reviewer viewed the work in an overall positive light, but requested and/or suggested further experimentation that will improve the data and their interpretation. I ask then, that you treat this as a 'major' revision, performing additional work where it is possible, and reasonable in terms of time; in which case the revised work should more easily and rapidly move through review during resubmission.*

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition, when you are ready to resubmit, please be prepared to provide the following:

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We are sorry that we cannot be more positive about your manuscript at this stage, but if you have any concerns or questions, please do not hesitate to contact us.

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Marco Vignuzzi

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Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In this study by McCarthy et al., the authors follow up on a published observation that infection with a pathogenic strain of CHIKV leads to an overall disorganization of the dLN including the failure of lymphocytes to accumulate and GCs to develop. Here, the authors perform a detailed, mechanistic analysis of what leads to this phenotype and show nicely that in mice infected with a “pathogenic” strain AF15561 there are increases in monocytes and neutrophils in the dLN early in infection and depletion of these cells leads to an accumulation of lymphocytes and restoration of the dLN organization after CHIKV infection. They go on to show that depletion of Gr-1 positive cells also increases CHIKV specific B-cell responses. To continue their mechanistic studies, they perform a beautiful set of genetic experiments to show that the monocyte recruitment is dependent on MyD88, Nos2 and Nox2 are involved in the disruption of the dLN during CHIKV infection, and iNos expression is controlled by an IFNAR and IRF5 dependent pathway. Finally, they show that there are distinct pathways activation ISGs in the footpad (IRF3/IRF7 dependent) and the dLN (IRF5 dependent). Overall, this study was well-written, easy to follow, and is novel, contributing to multiple areas of CHIKV infection. However, I had a few concerns that will should be addressed.

Reviewer #2: Previously, this group has shown that pathogenic CHIKV strains persist in the joint tissues of WT mice while the attenuated vaccine strain was cleared in a fashion dependent on virus-specific antibody responses. In this study, McCarthy et. al. identified two cell types, neutrophils and inflammatory monocytes, that when depleted increase lymphocyte accumulation and improve draining lymph node (dLN) organization and CHIKV-specific B cell responses prior to infection with a pathogenic and chronic CHIKV strain (AF15561). This is due to iNOS and NOX2 expression in a IRF5/IFNAR1-dependent and partially TLR7-dependent mechanism.

This work is important and novel in two main ways. The authors have 1) defined the cell types and pathways responsible for differential B cell responses to acute versus chronic CHIKV infections, and 2) identified differential innate immune gene expression programs in the site of inoculation versus dLN. This study has left some interesting remaining questions: 1) what are the MyD88-dependent pathways in addition to TLR7, and 2) what is the cell type requirement for IRF5? The manuscript is well-written, and the model is nicely developed and supported by data that demonstrates both rigor and statistical power. Overall, this is a very nice study. Only a few issues need to be addressed.

Reviewer #3: The report from McCarthy et al describes mechanisms behind differences in the control of pathogenic versus attenuated chikungunya virus strains. Multiple transgenic mouse strains are used in this detailed description of the events up- and downstream of the role monocytes and neutrophils play in disrupting effective B-cell responses in draining lymph nodes. This is an excellent, and very thorough study, likely to be of general interest and highly impactful for the field.

The differences between attenuated and pathogenic virus in terms of monocyte recruitment into draining LNs, the mechanisms behind this and the downstream consequences to LNs and germinal center formation are all clearly described and supported. The only criticism of the study is that although there are claims that these local innate immune responses “…..dictate the effectiveness of downstream adaptive immunity”, there is very little actual evidence to support this. There is no discussion as to whether and how these minimal changes may represent the stark differences in pathogenicity seen. More detailed time-courses, isotype differences and/or study of anti-CHIKV memory B-cells or long-lived plasma cells from outside the LN may also identify more significant differences. There is also no discussion of impact on disease outcomes in the mice. Obviously there are issues with downstream effects on viral clearance by these cells in tissues, however with careful timing, or in some of the mouse strains, it seems like some mitigation of disease should be observed if the germinal center disruption really plays a significant role in pathogenesis.

Despite this caveat, the study remains highly informative and will move the field forward significantly.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. The authors make the point several times that the clearance of attenuated, acute CHIKV infections are due to B-cell and Ab responses (Lines 80-81 for example) and that during persistent, pathogenic CHIKV infections “virus-specific B-cell responses may be impaired” (Lines 82-83). I took this as an important point of the paper and that the authors were hypothesizing that the impaired B cell response is what is responsible or partly responsible for persistence of pathogenic CHIKV. The authors do address B-cell specific responses in Figure 4 and nicely show increases in CHIKV specific B-cells. However, it is unclear if this increase in B cells leads to increased clearance of pathogenic CHIKV and is a potential mechanism for persistence. The authors should measure viral titers in Gr-1 depleted mice to determine if pathogenic CHIKV has increased clearance after monocyte depletion and B cell recruitment. If this was not the point of the paper the authors may want to focus the writing on the disorganized dLN, etc phenotype and leave out viral clearance and persistence until the discussion. If this was addressed but didn’t work, the authors should include some lines in the discussion about potential mechanisms.

2. In the title and their conclusions, the authors make the point that this MyD88-dependent recruitment of monocytes impairs CHIKV B-cell responses. However, the authors only show CHIKV specific B cells after Gr-1 treatment. What about in My88 deficient mice? Nos, Nox, and IRF5 deficient mice? The authors should address lymphocyte recruitment to dLN and CHIKV Abs in these mice.

3. The use of “attenuated” and “pathogenic, persistent” to describe the CHIKV strains was a bit confusing. There are other pathogenic strains of CHIKV that may not be persistent strains (IOL for example) and relative to each other, some strains may be considered “attenuated”. At this point, it is unclear if this same mechanism holds up for all of these strains and until it’s address I suggest calling these viruses as they are “vaccine” and “AF15561” or “parent”.

Reviewer #2: 1. It is not clear what the molecular mechanism and functional consequences of improved B cell responses are. In the previous study (Hawman et al, Cell Rep 2016), the pathogenic CHIKV strain AF15561 has evolved ways to evade neutralizing antibody responses against E2. How does myeloid cell depletion in AF15561-infected mice improve neutralization activity of serum? Has the virus mutated? Is this increase in serum neutralization activity sufficient to restrict viral replication in the joint tissues and facilitate viral clearance? It would be informative to perform passive transfer of serum from Gr-1 antibody treated mice and include more discussions on this data.

2. In Figure 1A, dLN from AF15561-infected mice has significantly more cells in general. What are these cells other than the neutrophils and monocytes? It would be informative to immunophenotype all the cells in the dLN. Since depletion of both neutrophil and monocytes does not completely rescue CD8+ T cells from the dLN (Fig. 2K) and IRF5 is required and constitutively expressed in DCs, it is possible that DCs might play a role in viral persistence. Does pathogenic CHIKV infection increase the influx of DCs into dLN?

Reviewer #3: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. Figure 1B: There is a * as significant that is not reflected in the figure legend.

2. Figure 7: The figure legend is swapped with the figure. For example, Figure 7F is described in what is B in the legend. Figure G-H are described in C-D of the legend.

Reviewer #2: 1. In a few occasions, the data does not fully support the model and the writing needs to be modified to take that into consideration. For example, in lines 209-210, the authors stated that “IL-1R-independent, MyD88-dependent signal(s) promote the recruitment of monocytes to the dLN”. However, monocyte number is significantly reduced in the dLN of IFNAR knockout mice although the percentage of monocytes is not affected, suggesting that IFN signaling might have an effect on monocyte accumulation. Additionally, most of the ISGs tested in Figure 8 demonstrate strict IRF5 dependency in dLN except for Ifit1 and Rsad2. Is it possible that the dichotomy of IRF3/7-dependent ISG expression near site of inoculation and IRF5-dependent ISG expression in dLN is gene specific?

2. Lines 27-28 should be changed to “Infection of WT mice with pathogenic but not acutely cleared CHIKV induced MyD88-dependent recruitment”.

3. In lines 189-190, the use of double negative is confusing and difficult to understand.

4. Line 237 should refer to Fig 6G.

Reviewer #3: Fig. 4L and M would be more informative with absolute levels demonstrated, rather than fold change which can be deceiving.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

6 Dec 2019

Submitted filename: McCarthy et al-Response to Reviewer Comments-FINAL.pdf

Click here for additional data file.

22 Dec 2019

Dear Dr. Morrison, dear Dr. McCarthy, and fellow collaborators,

We are pleased to inform that your manuscript, "MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2", has been editorially accepted for publication at PLOS Pathogens.

Before your manuscript can be formally accepted and sent to production, you will need to complete our formatting changes, which you will receive by email within a week. Please note that your manuscript will not be scheduled for publication until you have made the required changes.

IMPORTANT NOTES

(1) Please note, once your paper is accepted, an uncorrected proof of your manuscript will be published online ahead of the final version, unless you’ve already opted out via the online submission form. If, for any reason, you do not want an earlier version of your manuscript published online or are unsure if you have already indicated as such, please let the journal staff know immediately at plospathogens@plos.org.

(2) Copyediting and Proofreading: The corresponding author will receive a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript.

(3) Appropriate Figure Files: Please remove all name and figure # text from your figure files. Please also take this time to check that your figures are of high resolution, which will improve the readbility of your figures and help expedite your manuscript's publication. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plospathogens/s/figures.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

(4) Striking Image: Please upload a striking still image to accompany your article if one is available (you can include a new image or an existing one from within your manuscript). Should your paper be accepted, this image will be considered for our monthly issue image and may also appear on our website to feature your article. Please upload this as a separate file, selecting "striking image" as the file type upon upload. Please also include a separate "Other" file with a caption, including credits and any potential copyright information. Please do not include the caption in the main article file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plospathogens/s/content-license. Please note that PLOS cannot publish copyrighted images.

(5) Press Release or Related Media: If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team in advance at plospathogens@plos.org as soon as possible. We ask that you contact us within one week to plan ahead of our fast Production schedule. If you need to know your paper's publication date for related media purposes, you must coordinate with our press team, and your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version.

(6)  PLOS requires an ORCID iD for all corresponding authors on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.  To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field.  This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager

(7) Update your Profile Information: Now that your manuscript has been provisionally accepted, please log into Editorial Manager and update your profile, if needed. Go to https://www.editorialmanager.com/ppathogens, log in, and click on the "Update My Information" link at the top of the page. Please update your user information to ensure an efficient production and billing process.

(8) LaTeX users only: Our staff will ask you to upload a TEX file in addition to the PDF before the paper can be sent to typesetting, so please carefully review our Latex Guidelines http://journals.plos.org/plospathogens/s/latex in the meantime.

(9) If you have associated protocols in protocols.io, please ensure that you make them public before publication to guarantee immediate access to the methodological details.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Marco Vignuzzi

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Grant McFadden

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-2556-3526

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: I appreciate the author's efforts to address my comments and the other reviewer's concerns. I have no further concerns and am looking forward to the follow up studies.

Reviewer #2: The authors have nicely addressed all my concerns.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

22 Jan 2020

Dear Dr. Morrison,

We are delighted to inform you that your manuscript, "MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064