FcRL4 Expression Identifies a Pro-inflammatory B Cell Subset in Viremic HIV-Infected Subjects

In autoimmune diseases, toll-like receptor (TLR)-stimulated pro-inflammatory IL-6-secreting B cells exert pathogenic roles. Similarly, B cell Fc receptor-like 4 (FcRL4) expression amplifies TLR stimulation, and in rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B cell subset. B cells from HIV-infected subjects also express heightened levels of FcRL4 and secrete high levels of IL-6: a critical mediator of HIV disease progression. In this study, we sought to determine if FcRL4 identifies a pro-inflammatory B cell subset in HIV-infected subjects and further elucidate the mechanisms underlying FcRL4 amplification of TLR stimulation. We determine that tissue-like memory B cells express the highest endogenous levels of FcRL4 positively correlating with IL-6 expression (p = 0.0022, r = 0.8667), but activated memory B cells exhibit the highest frequency of FcRL4hiIL-6hi cells. FcRL4hi B cells exhibit an activated TLR-signaling pathway identified by elevated phosphorylation levels of: pERK (p = 0.0373), p38 (p = 0.0337), p65 (p = 0.1097), and cJUN (p = 0.0239), concomitant with significantly elevated expression of the TLR-signaling modulator hematopoietic cell kinase (HcK, p = 0.0414). Compared to FcRL4neg B cells from healthy controls, TLR9-stimulated FcRL4pos B cells express significantly higher levels of lL-6 (p = 0.0179). Further, TLR9-stimulated B cells also upregulate the expression of FcRL4 (p = 0.0415) and HcK (p = 0.0386). In B-cell lines, siRNA-mediated HcK knockdown downmodulates TLR9-induced FcRL4-mediated activation quantified by CD23 upregulation (p = 0.0553). We present data suggesting that, in viremic HIV-infected individuals, FcRL4 expression identifies unique IL-6 producing pro-inflammatory B-cell subsets. Further, TLR stimulation likely modulates FcRL4 expression and FcRL4 expression is associated with Hck, potentially enhancing the activation of TLR-signaling associated transcription factors. Pathogenic B-cells have been identified in other disease settings, and this study represents a novel report describing a pro-inflammatory B cell subset in HIV-infected patients.

In autoimmune diseases, toll-like receptor (TLR)-stimulated pro-inflammatory IL-6secreting B cells exert pathogenic roles. Similarly, B cell Fc receptor-like 4 (FcRL4) expression amplifies TLR stimulation, and in rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B cell subset. B cells from HIV-infected subjects also express heightened levels of FcRL4 and secrete high levels of IL-6: a critical mediator of HIV disease progression. In this study, we sought to determine if FcRL4 identifies a pro-inflammatory B cell subset in HIV-infected subjects and further elucidate the mechanisms underlying FcRL4 amplification of TLR stimulation. We determine that tissue-like memory B cells express the highest endogenous levels of FcRL4 positively correlating with IL-6 expression (p = 0.0022, r = 0.8667), but activated memory B cells exhibit the highest frequency of FcRL4 hi IL-6 hi cells. FcRL4 hi B cells exhibit an activated TLR-signaling pathway identified by elevated phosphorylation levels of: pERK (p = 0.0373), p38 (p = 0.0337), p65 (p = 0.1097), and cJUN (p = 0.0239), concomitant with significantly elevated expression of the TLR-signaling modulator hematopoietic cell kinase (HcK, p = 0.0414). Compared to FcRL4 neg B cells from healthy controls, TLR9-stimulated FcRL4 pos B cells express significantly higher levels of lL-6 (p = 0.0179). Further, TLR9-stimulated B cells also upregulate the expression of FcRL4 (p = 0.0415) and HcK (p = 0.0386). In B-cell lines, siRNA-mediated HcK knockdown downmodulates TLR9-induced FcRL4-mediated activation quantified by CD23 upregulation (p = 0.0553). We present data suggesting that, in viremic HIV-infected individuals, FcRL4 expression identifies unique IL-6 producing pro-inflammatory B-cell subsets. Further, TLR stimulation likely modulates FcRL4 expression and FcRL4 expression is associated with Hck, potentially enhancing the activation of TLR-signaling associated transcription factors. Pathogenic B-cells have been identified in other disease settings, and this study represents a novel report describing a pro-inflammatory B cell subset in HIV-infected patients. The elevated serum level of the pro-inflammatory cytokine IL-6 is an indicator of chronic immune activation and a driver of HIV disease progression (1,2). During HIV infection, IL-6 overexpression drives B-cell proliferation, enhances secretion of antibodies, and leads to aberrant B cell terminal differentiation (3,4). Further, in vitro, IL-6 has been shown to drive HIV replication and, in HIV-infected individuals, the observed high levels of IL-6 are associated with increased mortality and morbidity (5,6). Due to these factors, it is critical to determine the sources of IL-6 as well as the mechanisms underlying IL-6 overexpression during HIV infection. HIV infection is characterized by heightened microbial translocation and the presence of microbial products encoding toll-like receptor ligands (TLR-L) (7)(8)(9). TLR-stimulated monocytes have been identified to be a significant contributor to the HIV-induced inflammatory state (10)(11)(12). However, published data also suggest that B cells from HIV-infected individuals express high levels of IL-6 possibly due to TLR-stimulation (3,9,13). Additionally, in autoimmune diseases, TLR-stimulated B-cells are critical mediators of inflammation (14,15). Further, data from a study in rheumatoid arthritis identified a pro-inflammatory B-cell subset expressing high levels of Fc receptor-like 4 (FcRL4) (16). FcRL4 acts as a molecular switch, dampening B cell receptor (BCR) signaling while simultaneously enhancing TLR-signaling through association of SHP-1 and SHP-2 with its cytoplasmic tail (17). Finally, B cells from HIV-infected viremic subjects exhibit heightened FcRL4 expression associated with an "exhausted" phenotype, with impaired antibody expressing functions (18)(19)(20).
In this study, we investigated: (1) if in untreated HIV infection, FcRL4 hi B-cells represent a pro-inflammatory B cell subset and (2) the mechanisms underlying FcRL4 expression and amplification of TLR-signaling. Our data indicate that FcRL4 hi B-cell subsets are high producers of IL-6, and TLR-signaling modulates FcRL4 expression. Finally, FcRL4 mediates amplification of TLRsignaling likely by recruiting Src Kinase proteins.

MaTerials anD MeThODs study Participants
All studies were performed after signed, informed written research consent by each study subject. The study was reviewed and approved by the Institutional Review Board of the Rush University Medical Center, and the University of Iowa City VAMC and University of Iowa. All work was performed in adherence with appropriate laboratory safety protocols such as use of personal protective equipment. HIV-infected viremic (HIVVIR), naïve subjects had a median CD4 count of 466 cells/μl (range, 144-566), and median viral load of 20,000 copies/ml (range, 2,000-117,000) ( Table 1).

cell lines
Ramos (a human Burkitt lymphoma cell line) FcRL4 stable transfectants were a generous gift from Dr. Susan Pierce (NIH) and previously described (17). The FcRL4.

inhibition assays
Chemical inhibition of Hck was achieved using PP2 (Millipore). Cells were incubated overnight with indicated concentrations of the inhibitor, supplemented with TLR9-L, and further cultured overnight. Only events corresponding to living cells (determined by Live/Dead ® Fixable Aqua staining, Life Technologies) were acquired on an LRSII (BD Biosciences) flow cytometer and the data analyzed using FlowJo software (Tree Star Inc.).

real-time rT-Pcr
RNA was extracted using the RNeasy Kit (QIAGEN) according to the manufacturer's instructions. The extracted RNA was measured by spectrophotometer and equimolar concentrations used for cDNA synthesis according to the manufacturer's instructions (iScript cDNA syntesis Kit, Bio-Rad). The following primers were used for the qPCR reaction: HcK-Forward 5′-CGGATCCCACATCCACCATCA-3′, Reverse 5′-ACCACGA TGATGTCCTCAGAGC-3′, FcRL4-Forward 5′-TCAGCTGGG AGAAGAAGAGGAA-3′, Reverse 5′-GAGTTATCTGGGTGTT GTGTCTTTACC-3′, GAPDH-Forward 5′-CTTCAACGACCA CTTTGT-3′ and reverse 5′-TGGTCCAGGGGTCTTACT-3′. Real-time RT-PCR was performed using a Quantitect SYBR Green PCR kit (Qiagen) in a 7900HT Fast Real-Time PCR system (Applied Biosystems). Melting curve analysis was performed to ensure that the primers amplified the desired amplicon and that primer-dimers were absent. Fold change in mRNA expression was calculated by relative quantification using the comparative cycle threshold method. GAPDH expression was used as an endogenous control.

sirna-Mediated Knockdown
siRNA targeting HcK were purchased from Santa Cruz Biotechnology and Dharmacon, and cells were transfected using the Lipofectamine RNAiMax kit (Life Technologies) according to the manufacturer's instructions. Knockdown was confirmed by qPCR 48H post-transfection.

statistical analysis
Results are expressed as mean ± SEM or as indicated. GraphPad Prism software, version 5.03 was used for all statistical analysis. The statistical significance p-value between group parameters was determined using either unpaired or paired Student's t-test (with a confidence level of 95%). The statistical dependence between variables was calculated using the Spearman rank correlation analysis. p-Values of <0.05 were considered statistically significant. Pair and multiple comparisons were done using the Wilcoxon-matched-pairs signed rank test.

Fcrl4 pos B cells from hiV-infected Viremic (hiV Vir ) subjects constitutively exhibit an activated Tlr-signaling cascade
HIV-infection is associated with an increase in serum concentration of several TLR ligands (7)(8)(9), and B cells from HIVVIR individuals exhibit enhanced FcRL4 expression (18). As FcRL4 enhances B-cell responsiveness to TLR stimulation (17), we next investigated if, in HIVVIR subjects, constitutive FcRL4 expression is associated with an activated TLR-signaling pathway. We determined that FcRL4 pos B cells of HIVVIR subjects exhibit a constitutively activated TLR-signaling pathway phenotype characterized by significantly elevated levels of phosphorylated ERK, p38, and c-JUN (Figures 2A,B, p = 0.0373, p = 0.0337, and p = 0.0239, respectively). Although the level of phosphorylated p65 was higher in FcRL4 pos B cells than FcRL4 neg B cells, the difference did not attain statistical significance (Figure 2B, p = 0.1097).

Fcrl4 pos B cells from hiV-Uninfected subjects are highly responsive to Tlr stimulation
We previously demonstrated that TLR stimulated B cells from healthy controls (HIVNEG) subjects upregulate expression of the pro-inflammatory cytokine IL-6 (9). We, therefore, examined if FcRL4 modulates the expression of IL-6 upon TLR stimulation. We found that compared to FcRL4-negative (FcRL4 neg ) B cells, TLR stimulation of purified FcRL4 pos B cells significantly upregulated IL-6 expression (Figure 3: TLR2, p = 0.0022, TLR7, p = 0.0286, TLR9, p = 0.0179).  B cells exposed to Tlr-9 ligand Upregulate expression of Fcrl4 and hcK concomitantly Elevated FcRL4 expression on blood B cells has been identified in malaria and HIV-infected viremic patients (18,19), conditions associated with heightened serum levels of TLR ligands (7,8,21). Additionally, it has been previously demonstrated that TLR stimulation modulates FcRL expression in mice (22). We determined that exposure of PBMC from HIVNEG subjects to TLR9 stimulation led to a significant upregulation in FcRL4 expression ( Figure 4A, p = 0.0415). We confirmed that while TLR stimulation induces FcRL4 upregulation, the anti-FcRL4 flow cytometry antibody did not lead to FcRL4 upregulation. In human secondary lymphoid tissue, elevated FcRL4 expression is associated with heightened levels of the Src kinase family member HcK (23), which in macrophages, promotes TLR-induced expression of pro-inflammatory cytokines (24). We, therefore, investigated if in TLR-stimulated blood B cells, the observed FcRL4 upregulation ( Figure 4A) is associated with heightened HcK expression contributing to the amplification of the TLR-signaling. We determined that TLR9-stimulation of purified B cells from HIVNEG resulted in the upregulation of HcK levels ( Figure 4B, p = 0.0386) (gating Figure S2 in Supplementary Material). Finally, in HIVVIR subjects, FcRL4 pos B cells, expressed significantly higher endogenous levels of total ( Figure 4C, p = 0.0414) and phosphorylated HcK ( Figure 4C, p = 0.0398).

DiscUssiOn
In this study, we demonstrate that during viremic HIV infection, FcRL4 hi TLM and AM blood B cells express high endogenous levels of IL-6, strongly indicating that high FcRL4 expression identifies pro-inflammatory B cells. We demonstrate that frequency of FcRL4 + B-cells correlates strongly with IL-6 + B-cell frequency in the TLM subset. However, AM B cells exhibit the highest frequency of FcRL4 + IL-6 + double-positive cells suggesting the possibility that divergent mechanisms drive IL-6 production in AM and TLM B cells. This concept of divergent mechanisms is further supported by the distinct characteristics of these subsets, with TLM displaying elevated expression of inhibitory receptors and increased frequency of HIV-specific B cells, while the AM subset show greater specificity for other pathogens (20). Taken together, our report identifies pro-inflammatory functions of FcRL4 + TLM B cells in viremic HIV-infected subjects, corroborating findings, which identify FcRL4 hi B cells as a marker of pro-inflammatory B cells in rheumatoid arthritis patients (16). Though FcRL4 has previously been identified on exhausted B-cell subsets (20), weak proliferation following BCR stimulation may be indicative of a shift in function rather than a general failure to respond. FcRL4 has been identified as a molecular switch, dampening BCR signaling while enhancing B-cell responsiveness to TLR-stimulation (17). HIV-infected viremic (HIVVIR) subjects exhibit elevated serum levels of TLR-ligands (7-9) concomitant with high expression of FcRL4 on B cells (18,20). It is, therefore, tempting to suggest that in HIVVIR subjects, TLM and AM B cells are stimulated by TLR-ligands resulting in upregulated FcRL4 expression. This increases sensitivity to TLR stimulation, leading to a positive feedback loop culminating in high expression of IL-6, inflammation, and HIV disease progression. Though we cannot exclude the possibility that FcRL4-expressing B cells coincidently express IL-6, our data provide further evidence supporting a role for FcRL4 in mediating in vivo TLR-signaling-dependent hyperstimulation during HIV infection. We also determined that ex vivo, FcRL4 hi B cells from HIVVIR subjects exhibit a TLR-signaling signature, characterized by heightened activation of NF-κB and AP1 pathways, transcription factors critical for the expression of pro-inflammatory genes (27)(28)(29).
While FcRL4 expression has been well documented in HIV, its function remains only partly elucidated. During HIV-1 infection, FcRL4 is elevated on TLM of non-treated individuals, but expression is greatly reduced following treatment (30); this suggests a unique role for FcRL4 during HIV infection. Jelicic et al. report that HIV gp120 induces FcRL4 expression on B cells (31), suggesting another mechanism inducing FcRL4 expression, which enhances susceptibility to TLR stimulation in HIV infection. Previous studies also suggest that another FcRL family protein, FcRL3, is upregulated in response to TLR stimulation (32); however, a role for TLR stimulation in regulating FcRL4 expression in HIV infection has not been explored. We provide data suggesting that TLR-signaling augments B-cell FcRL4 expression, corroborating reports of TLR-regulation of FcRL3 (32). Though we present data indicating B cells exposed to TLR9ligand CpG-ODN2006 upregulate FcRL4 expression, we also observed comparable effects when B cells are exposed to either TLR7 (Imiquimod) or TLR2 (Pam3Csk4) ligands (not shown).   (14), and some reports also suggest that during HIV infection B-cells express IL-6, thus  likely exerting a pathogenic role (3,9,13). Our data present FcRL4 as a marker identifying potential pro-inflammatory B cells during viremic HIV infection.

cOnclUsiOn
The data from this study indicate that in viremic HIV infected subjects, high expression of FcRL4 identifies pro-inflammatory B cell subsets. In autoimmune conditions, B cells have been established as critical IL-6 expressing cells (16). Our data demonstrate a pro-inflammatory function of FcRL4 + B cells, a population of B cells previously identified as exhausted, in viremic HIV infection. Finally, we present data elucidating the mechanisms of FcRL4-mediated amplification of TLRsignaling in B cells. We provide data indicating that increased expression of FcRL4 coincides with upregulation of the Src kinase HcK, and HcK is necessary for FcRL4's amplification of TLR signaling.

eThics sTaTeMenT
All studies were performed after signed informed written research consent by each study subject.